HNRNPC haploinsufficiency affects alternative splicing of intellectual disability-associated genes and causes a neurodevelopmental disorder.

Heterogeneous nuclear ribonucleoprotein C (HNRNPC) is an essential, ubiquitously abundant protein involved in mRNA processing. Genetic variants in other members of the HNRNP family have been associated with neurodevelopmental disorders. Here, we describe 13 individuals with global developmental delay, intellectual disability, behavioral abnormalities, and subtle facial dysmorphology with heterozygous HNRNPC germline variants. Five of them bear an identical in-frame deletion of nine amino acids in the extreme C terminus. To study the effect of this recurrent variant as well as HNRNPC haploinsufficiency, we used induced pluripotent stem cells (iPSCs) and fibroblasts obtained from affected individuals. While protein localization and oligomerization were unaffected by the recurrent C-terminal deletion variant, total HNRNPC levels were decreased. Previously, reduced HNRNPC levels have been associated with changes in alternative splicing. Therefore, we performed a meta-analysis on published RNA-seq datasets of three different cell lines to identify a ubiquitous HNRNPC-dependent signature of alternative spliced exons. The identified signature was not only confirmed in fibroblasts obtained from an affected individual but also showed a significant enrichment for genes associated with intellectual disability. Hence, we assessed the effect of decreased and increased levels of HNRNPC on neuronal arborization and neuronal migration and found that either condition affects neuronal function. Taken together, our data indicate that HNRNPC haploinsufficiency affects alternative splicing of multiple intellectual disability-associated genes and that the developing brain is sensitive to aberrant levels of HNRNPC. Hence, our data strongly support the inclusion of HNRNPC to the family of HNRNP-related neurodevelopmental disorders.

Web-accessible application for identifying pathogenic transcripts with RNA-seq: Increased sensitivity in diagnosis of neurodevelopmental disorders.

For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. Transcriptome analysis (RNA sequencing [RNA-seq]) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals' cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual's specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics.

Heterozygous ANKRD17 loss-of-function variants cause a syndrome with intellectual disability, speech delay, and dysmorphism.

ANKRD17 is an ankyrin repeat-containing protein thought to play a role in cell cycle progression, whose ortholog in Drosophila functions in the Hippo pathway as a co-factor of Yorkie. Here, we delineate a neurodevelopmental disorder caused by de novo heterozygous ANKRD17 variants. The mutational spectrum of this cohort of 34 individuals from 32 families is highly suggestive of haploinsufficiency as the underlying mechanism of disease, with 21 truncating or essential splice site variants, 9 missense variants, 1 in-frame insertion-deletion, and 1 microdeletion (1.16 Mb). Consequently, our data indicate that loss of ANKRD17 is likely the main cause of phenotypes previously associated with large multi-gene chromosomal aberrations of the 4q13.3 region. Protein modeling suggests that most of the missense variants disrupt the stability of the ankyrin repeats through alteration of core structural residues. The major phenotypic characteristic of our cohort is a variable degree of developmental delay/intellectual disability, particularly affecting speech, while additional features include growth failure, feeding difficulties, non-specific MRI abnormalities, epilepsy and/or abnormal EEG, predisposition to recurrent infections (mostly bacterial), ophthalmological abnormalities, gait/balance disturbance, and joint hypermobility. Moreover, many individuals shared similar dysmorphic facial features. Analysis of single-cell RNA-seq data from the developing human telencephalon indicated ANKRD17 expression at multiple stages of neurogenesis, adding further evidence to the assertion that damaging ANKRD17 variants cause a neurodevelopmental disorder.

SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission.

SLC4A10 is a plasma-membrane bound transporter that utilizes the Na+ gradient to drive cellular HCO3- uptake, thus mediating acid extrusion. In the mammalian brain, SLC4A10 is expressed in principal neurons and interneurons, as well as in epithelial cells of the choroid plexus, the organ regulating the production of CSF. Using next generation sequencing on samples from five unrelated families encompassing nine affected individuals, we show that biallelic SLC4A10 loss-of-function variants cause a clinically recognizable neurodevelopmental disorder in humans. The cardinal clinical features of the condition include hypotonia in infancy, delayed psychomotor development across all domains and intellectual impairment. Affected individuals commonly display traits associated with autistic spectrum disorder including anxiety, hyperactivity and stereotyped movements. In two cases isolated episodes of seizures were reported in the first few years of life, and a further affected child displayed bitemporal epileptogenic discharges on EEG without overt clinical seizures. While occipitofrontal circumference was reported to be normal at birth, progressive postnatal microcephaly evolved in 7 out of 10 affected individuals. Neuroradiological features included a relative preservation of brain volume compared to occipitofrontal circumference, characteristic narrow sometimes 'slit-like' lateral ventricles and corpus callosum abnormalities. Slc4a10 -/- mice, deficient for SLC4A10, also display small lateral brain ventricles and mild behavioural abnormalities including delayed habituation and alterations in the two-object novel object recognition task. Collapsed brain ventricles in both Slc4a10-/- mice and affected individuals suggest an important role of SLC4A10 in the production of the CSF. However, it is notable that despite diverse roles of the CSF in the developing and adult brain, the cortex of Slc4a10-/- mice appears grossly intact. Co-staining with synaptic markers revealed that in neurons, SLC4A10 localizes to inhibitory, but not excitatory, presynapses. These findings are supported by our functional studies, which show the release of the inhibitory neurotransmitter GABA is compromised in Slc4a10-/- mice, while the release of the excitatory neurotransmitter glutamate is preserved. Manipulation of intracellular pH partially rescues GABA release. Together our studies define a novel neurodevelopmental disorder associated with biallelic pathogenic variants in SLC4A10 and highlight the importance of further analyses of the consequences of SLC4A10 loss-of-function for brain development, synaptic transmission and network properties.

Exome sequencing in fetuses with congenital diaphragmatic hernia in a nationwide cohort.

OBJECTIVE: To evaluate the diagnostic yield of exome sequencing (ES) in fetuses and neonates with prenatally detected congenital diaphragmatic hernia (CDH) and normal copy number variant (CNV) analysis. METHODS: We conducted a retrospective cohort study of prenatally diagnosed CDH cases seen between 2019 and 2022. All cases who underwent prenatal or postnatal genetic testing were reviewed. The results from the ES analysis that identified pathogenic or likely pathogenic single nucleotide variants are described. RESULTS: In total, 133 fetuses with CDH were seen, of whom 98 (74%) had an isolated CDH and 35 (26%) had a complex CDH (associated structural anomalies) on prenatal examination. ES was performed in 68 cases, and eight pathogenic or likely pathogenic variants were found, accounting for a 12% diagnostic yield (10% [5/50] in isolated cases and 17% [3/18] in complex CDH). CONCLUSIONS: In 12% of fetuses and neonates with CDH and normal CNV analysis results, pathogenic or likely pathogenic variants were identified with ES. These data indicate that there is a substantial diagnostic yield when offering ES in prenatally detected CDH, both in complex and isolated cases.

Overlapping cortical malformations in patients with pathogenic variants in GRIN1 and GRIN2B.

BACKGROUND: Malformations of cortical development (MCDs) have been reported in a subset of patients with pathogenic heterozygous variants in GRIN1 or GRIN2B, genes which encode for subunits of the N-methyl-D-aspartate receptor (NMDAR). The aim of this study was to further define the phenotypic spectrum of NMDAR-related MCDs. METHODS: We report the clinical, radiological and molecular features of 7 new patients and review data on 18 previously reported individuals with NMDAR-related MCDs. Neuropathological findings for two individuals with heterozygous variants in GRIN1 are presented. We report the clinical and neuropathological features of one additional individual with homozygous pathogenic variants in GRIN1. RESULTS: Heterozygous variants in GRIN1 and GRIN2B were associated with overlapping severe clinical and imaging features, including global developmental delay, epilepsy, diffuse dysgyria, dysmorphic basal ganglia and hippocampi. Neuropathological examination in two fetuses with heterozygous GRIN1 variants suggests that proliferation as well as radial and tangential neuronal migration are impaired. In addition, we show that neuronal migration is also impaired by homozygous GRIN1 variants in an individual with microcephaly with simplified gyral pattern. CONCLUSION: These findings expand our understanding of the clinical and imaging features of the 'NMDARopathy' spectrum and contribute to our understanding of the likely underlying pathogenic mechanisms leading to MCD in these patients.

De novo TRPV4 Leu619Pro variant causes a new channelopathy characterised by giant cell lesions of the jaws and skull, skeletal abnormalities and polyneuropathy.

BACKGROUND: Pathogenic germline variants in Transient Receptor Potential Vanilloid 4 Cation Channel (TRPV4) lead to channelopathies, which are phenotypically diverse and heterogeneous disorders grossly divided in neuromuscular disorders and skeletal dysplasia. We recently reported in sporadic giant cell lesions of the jaws (GCLJs) novel, somatic, heterozygous, gain-of-function mutations in TRPV4, at Met713. METHODS: Here we report two unrelated women with a de novo germline p.Leu619Pro TRPV4 variant and an overlapping systemic disorder affecting all organs individually described in TRPV4 channelopathies. RESULTS: From an early age, both patients had several lesions of the nervous system including progressive polyneuropathy, and multiple aggressive giant cell-rich lesions of the jaws and craniofacial/skull bones, and other skeletal lesions. One patient had a relatively milder disease phenotype possibly due to postzygotic somatic mosaicism. Indeed, the TRPV4 p.Leu619Pro variant was present at a lower frequency (variant allele frequency (VAF)=21.6%) than expected for a heterozygous variant as seen in the other proband, and showed variable regional frequency in the GCLJ (VAF ranging from 42% to 10%). In silico structural analysis suggests that the gain-of-function p.Leu619Pro alters the ion channel activity leading to constitutive ion leakage. CONCLUSION: Our findings define a novel polysystemic syndrome due to germline TRPV4 p.Leu619Pro and further extend the spectrum of TRPV4 channelopathies. They further highlight the convergence of TRPV4 mutations on different organ systems leading to complex phenotypes which are further mitigated by possible post-zygotic mosaicism. Treatment of this disorder is challenging, and surgical intervention of the GCLJ worsens the lesions, suggesting the future use of MEK inhibitors and TRPV4 antagonists as therapeutic modalities for unmet clinical needs.

Cysteinyl-tRNA Synthetase Mutations Cause a Multi-System, Recessive Disease That Includes Microcephaly, Developmental Delay, and Brittle Hair and Nails.

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes responsible for charging tRNA molecules with cognate amino acids. Consistent with the essential function and ubiquitous expression of ARSs, mutations in 32 of the 37 ARS-encoding loci cause severe, early-onset recessive phenotypes. Previous genetic and functional data suggest a loss-of-function mechanism; however, our understanding of the allelic and locus heterogeneity of ARS-related disease is incomplete. Cysteinyl-tRNA synthetase (CARS) encodes the enzyme that charges tRNACys with cysteine in the cytoplasm. To date, CARS variants have not been implicated in any human disease phenotype. Here, we report on four subjects from three families with complex syndromes that include microcephaly, developmental delay, and brittle hair and nails. Each affected person carries bi-allelic CARS variants: one individual is compound heterozygous for c.1138C>T (p.Gln380∗) and c.1022G>A (p.Arg341His), two related individuals are compound heterozygous for c.1076C>T (p.Ser359Leu) and c.1199T>A (p.Leu400Gln), and one individual is homozygous for c.2061dup (p.Ser688Glnfs∗2). Measurement of protein abundance, yeast complementation assays, and assessments of tRNA charging indicate that each CARS variant causes a loss-of-function effect. Compared to subjects with previously reported ARS-related diseases, individuals with bi-allelic CARS variants are unique in presenting with a brittle-hair-and-nail phenotype, which most likely reflects the high cysteine content in human keratins. In sum, our efforts implicate CARS variants in human inherited disease, expand the locus and clinical heterogeneity of ARS-related clinical phenotypes, and further support impaired tRNA charging as the primary mechanism of recessive ARS-related disease.

Loss of SMPD4 Causes a Developmental Disorder Characterized by Microcephaly and Congenital Arthrogryposis.

Sphingomyelinases generate ceramide from sphingomyelin as a second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. Children from 12 unrelated families presented with microcephaly, simplified gyral pattern of the cortex, hypomyelination, cerebellar hypoplasia, congenital arthrogryposis, and early fetal/postnatal demise. Genomic analysis revealed bi-allelic loss-of-function variants in SMPD4, coding for the neutral sphingomyelinase-3 (nSMase-3/SMPD4). Overexpression of human Myc-tagged SMPD4 showed localization both to the outer nuclear envelope and the ER and additionally revealed interactions with several nuclear pore complex proteins by proteomics analysis. Fibroblasts from affected individuals showed ER cisternae abnormalities, suspected for increased autophagy, and were more susceptible to apoptosis under stress conditions, while treatment with siSMPD4 caused delayed cell cycle progression. Our data show that SMPD4 links homeostasis of membrane sphingolipids to cell fate by regulating the cross-talk between the ER and the outer nuclear envelope, while its loss reveals a pathogenic mechanism in microcephaly.

Integration of metabolomics with genomics: Metabolic gene prioritization using metabolomics data and genomic variant (CADD) scores.

The integration of metabolomics data with sequencing data is a key step towards improving the diagnostic process for finding the disease-causing genetic variant(s) in patients suspected of having an inborn error of metabolism (IEM). The measured metabolite levels could provide additional phenotypical evidence to elucidate the degree of pathogenicity for variants found in genes associated with metabolic processes. We present a computational approach, called Reafect, that calculates for each reaction in a metabolic pathway a score indicating whether that reaction is deficient or not. When calculating this score, Reafect takes multiple factors into account: the magnitude and sign of alterations in the metabolite levels, the reaction distances between metabolites and reactions in the pathway, and the biochemical directionality of the reactions. We applied Reafect to untargeted metabolomics data of 72 patient samples with a known IEM and found that in 81% of the cases the correct deficient enzyme was ranked within the top 5% of all considered enzyme deficiencies. Next, we integrated Reafect with Combined Annotation Dependent Depletion (CADD) scores (a measure for gene variant deleteriousness) and ranked the metabolic genes of 27 IEM patients. We observed that this integrated approach significantly improved the prioritization of the genes containing the disease-causing variant when compared with the two approaches individually. For 15/27 IEM patients the correct affected gene was ranked within the top 0.25% of the set of potentially affected genes. Together, our findings suggest that metabolomics data improves the identification of affected genes in patients suffering from IEM.

MACF1 Mutations Encoding Highly Conserved Zinc-Binding Residues of the GAR Domain Cause Defects in Neuronal Migration and Axon Guidance.

To date, mutations in 15 actin- or microtubule-associated genes have been associated with the cortical malformation lissencephaly and variable brainstem hypoplasia. During a multicenter review, we recognized a rare lissencephaly variant with a complex brainstem malformation in three unrelated children. We searched our large brain-malformation databases and found another five children with this malformation (as well as one with a less severe variant), analyzed available whole-exome or -genome sequencing data, and tested ciliogenesis in two affected individuals. The brain malformation comprised posterior predominant lissencephaly and midline crossing defects consisting of absent anterior commissure and a striking W-shaped brainstem malformation caused by small or absent pontine crossing fibers. We discovered heterozygous de novo missense variants or an in-frame deletion involving highly conserved zinc-binding residues within the GAR domain of MACF1 in the first eight subjects. We studied cilium formation and found a higher proportion of mutant cells with short cilia than of control cells with short cilia. A ninth child had similar lissencephaly but only subtle brainstem dysplasia associated with a heterozygous de novo missense variant in the spectrin repeat domain of MACF1. Thus, we report variants of the microtubule-binding GAR domain of MACF1 as the cause of a distinctive and most likely pathognomonic brain malformation. A gain-of-function or dominant-negative mechanism appears likely given that many heterozygous mutations leading to protein truncation are included in the ExAC Browser. However, three de novo variants in MACF1 have been observed in large schizophrenia cohorts.

Mucolipidosis type II and type III: a systematic review of 843 published cases.

PURPOSE: Mucolipidosis (ML) II, MLIII alpha/beta, and MLIII gamma are rare autosomal recessive lysosomal storage disorders. Data on the natural course of the diseases are scarce. These data are important for counseling, therapies development, and improvement of outcome. The aim of this study is to gain knowledge on the natural history of ML by obtaining data on survival, symptom onset, presenting symptoms, diagnosis, and pathogenic variants associated with the MLII or MLIII phenotype. METHODS: A systematic review on all published MLII and MLIII cases between 1968 and August 2019 was performed. RESULTS: Three hundred one articles provided data on 843 patients. Median age at diagnosis: 0.7 for MLII and 9.0 years for MLIII. Median survival: 5.0 for MLII and 62.0 years for MLIIIII. Median age of death: 1.8 for MLII and 33.0 years for MLIII. Most frequent causes of death in all ML were pulmonary and/or cardiac complications. Pathogenic variants were described in 388 patients (GNPTAB: 571, GNPTG 179). CONCLUSION: This review provides unique insights into the natural history of MLII and MLIII, with a clear genotype-phenotype correlation with the most frequent pathogenic variant c.3503_3504del in MLII and in MLIII alpha/beta c.22A>G for GNPTAB. All pathogenic GNPTG variants resulted in MLIII gamma.

The potential diagnostic yield of whole exome sequencing in pregnancies complicated by fetal ultrasound anomalies.

INTRODUCTION: The aim of this retrospective cohort study was to determine the potential diagnostic yield of prenatal whole exome sequencing in fetuses with structural anomalies on expert ultrasound scans and normal chromosomal microarray results. MATERIAL AND METHODS: In the period 2013-2016, 391 pregnant women with fetal ultrasound anomalies who received normal chromosomal microarray results, were referred for additional genetic counseling and opted for additional molecular testing pre- and/or postnatally. Most of the couples received only a targeted molecular test and in 159 cases (40.7%) whole exome sequencing (broad gene panels or open exome) was performed. The results of these molecular tests were evaluated retrospectively, regardless of the time of the genetic diagnosis (prenatal or postnatal). RESULTS: In 76 of 391 fetuses (19.4%, 95% CI 15.8%-23.6%) molecular testing provided a genetic diagnosis with identification of (likely) pathogenic variants. In the majority of cases (91.1%, 73/76) the (likely) pathogenic variant would be detected by prenatal whole exome sequencing analysis. CONCLUSIONS: Our retrospective cohort study shows that prenatal whole exome sequencing, if offered by a clinical geneticist, in addition to chromosomal microarray, would notably increase the diagnostic yield in fetuses with ultrasound anomalies and would allow early diagnosis of a genetic disorder irrespective of the (incomplete) fetal phenotype.

Goldberg-Shprintzen syndrome is determined by the absence, or reduced expression levels, of KIFBP.

Goldberg-Shprintzen syndrome (GOSHS) is caused by loss of function variants in the kinesin binding protein gene (KIFBP). However, the phenotypic range of this syndrome is wide, indicating that other factors may play a role. To date, 37 patients with GOSHS have been reported. Here, we document nine new patients with variants in KIFBP: seven with nonsense variants and two with missense variants. To our knowledge, this is the first time that missense variants have been reported in GOSHS. We functionally investigated the effect of the variants identified, in an attempt to find a genotype-phenotype correlation. We also determined whether common Hirschsprung disease (HSCR)-associated single nucleotide polymorphisms (SNPs), could explain the presence of HSCR in GOSHS. Our results showed that the missense variants led to reduced expression of KIFBP, while the truncating variants resulted in lack of protein. However, no correlation was found between the severity of GOSHS and the location of the variants. We were also unable to find a correlation between common HSCR-associated SNPs, and HSCR development in GOSHS. In conclusion, we show that reduced, as well as lack of KIFBP expression can lead to GOSHS, and our results suggest that a threshold expression of KIFBP may modulate phenotypic variability of the disease.

Heterogeneous clinical phenotypes and cerebral malformations reflected by rotatin cellular dynamics.

Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects.

SYT1-associated neurodevelopmental disorder: a case series.

Synaptotagmin 1 (SYT1) is a critical mediator of fast, synchronous, calcium-dependent neurotransmitter release and also modulates synaptic vesicle endocytosis. This paper describes 11 patients with de novo heterozygous missense mutations in SYT1. All mutations alter highly conserved residues, and cluster in two regions of the SYT1 C2B domain at positions Met303 (M303K), Asp304 (D304G), Asp366 (D366E), Ile368 (I368T) and Asn371 (N371K). Phenotypic features include infantile hypotonia, congenital ophthalmic abnormalities, childhood-onset hyperkinetic movement disorders, motor stereotypies, and developmental delay varying in severity from moderate to profound. Behavioural characteristics include sleep disturbance and episodic agitation. Absence of epileptic seizures and normal orbitofrontal head circumference are important negative features. Structural MRI is unremarkable but EEG disturbance is universal, characterized by intermittent low frequency high amplitude oscillations. The functional impact of these five de novo SYT1 mutations has been assessed by expressing rat SYT1 protein containing the equivalent human variants in wild-type mouse primary hippocampal cultures. All mutant forms of SYT1 were expressed at levels approximately equal to endogenous wild-type protein, and correctly localized to nerve terminals at rest, except for SYT1M303K, which was expressed at a lower level and failed to localize at nerve terminals. Following stimulation, SYT1I368T and SYT1N371K relocalized to nerve terminals at least as efficiently as wild-type SYT1. However, SYT1D304G and SYT1D366E failed to relocalize to nerve terminals following stimulation, indicative of impairments in endocytic retrieval and trafficking of SYT1. In addition, the presence of SYT1 variants at nerve terminals induced a slowing of exocytic rate following sustained action potential stimulation. The extent of disturbance to synaptic vesicle kinetics is mirrored by the severity of the affected individuals' phenotypes, suggesting that the efficiency of SYT1-mediated neurotransmitter release is critical to cognitive development. In summary, de novo dominant SYT1 missense mutations are associated with a recognizable neurodevelopmental syndrome, and further cases can now be diagnosed based on clinical features, electrophysiological signature and mutation characteristics. Variation in phenotype severity may reflect mutation-specific impact on the diverse physiological functions of SYT1.

EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions: State of the art 2023.

Testing for AZoospermia Factor (AZF) deletions of the Y chromosome is a key component of the diagnostic workup of azoospermic and severely oligozoospermic men. This revision of the 2013 European Academy of Andrology (EAA) and EMQN CIC (previously known as the European Molecular Genetics Quality Network) laboratory guidelines summarizes recent clinically relevant advances and provides an update on the results of the external quality assessment program jointly offered by both organizations. A basic multiplex PCR reaction followed by a deletion extension analysis remains the gold-standard methodology to detect and correctly interpret AZF deletions. Recent data have led to an update of the sY84 reverse primer sequence, as well as to a refinement of what were previously considered as interchangeable border markers for AZFa and AZFb deletion breakpoints. More specifically, sY83 and sY143 are no longer recommended for the deletion extension analysis, leaving sY1064 and sY1192, respectively, as first-choice markers. Despite the transition, currently underway in several countries, toward a diagnosis based on certified kits, it should be noted that many of these commercial products are not recommended due to an unnecessarily high number of tested markers, and none of those currently available are, to the best of our knowledge, in accordance with the new first-choice markers for the deletion extension analysis. The gr/gr partial AZFc deletion remains a population-specific risk factor for impaired sperm production and a predisposing factor for testicular germ cell tumors. Testing for this deletion type is, as before, left at the discretion of the diagnostic labs and referring clinicians. Annual participation in an external quality control program is strongly encouraged, as the 22-year experience of the EMQN/EAA scheme clearly demonstrates a steep decline in diagnostic errors and an improvement in reporting practice.

5,10-methenyltetrahydrofolate synthetase deficiency: An extreme rare defect of folate metabolism in two Dutch siblings.

Two siblings, presenting with a neurometabolic phenotype, were identified with 5, 10-methenyltetrahydrofolate synthetase (MTHFS) deficiency. Whole genome sequencing in both patients demonstrated an homozygous MTHFS variant NM_006441.3(MTHFS):c.434G > A, p.Arg145Gin, which has been described before. At baseline, both patients showed moderate hyperhomocysteinemia, decreased 5-methyltetrahydrofolate (5MTHF), and increased 5-formyltetrahydrofolate (5-FTHF) in whole blood. In CSF, 5MTHF levels were in the low-normal range and 5-FTHF was strongly increased. In our novel enzyme assay, MTHFS activity was deficient in cultured fibroblasts in both sisters. Oral treatment was initiated with escalating dose of 5-methyltetrahydrofolate (5MTHF) up to 12 mg and hydroxycobalamin 5 mg daily. Plasma homocysteine normalized and 5MTHF became elevated in the blood of both patients. The elevated 5FTHF levels increased further on treatment in blood and CSF. This regimen resulted in some clinical improvement of patient 1. In patient 2, the clinical benefits of 5MTHF supplementation were less obvious. It seems plausible that the alleviation of the deficient 5MTHF levels and normalization of homocysteine in blood are of some clinical benefit. On the other hand, the very high levels of 5FTHF may well be detrimental and may prompt us to decrease the dose of 5MTHF. In addition, we hypothesize that the crippled MTHFS enzyme may destabilize the purinosome, which is presumably not ameliorated by 5MTHF.

Confirmation and expansion of the phenotype of the TCEAL1-related neurodevelopmental disorder.

Numerous contiguous gene deletion syndromes causing neurodevelopmental disorders have previously been defined using cytogenetics for which only in the current genomic era the disease-causing genes have become elucidated. One such example is deletion at Xq22.2, previously associated with a neurodevelopmental disorder which has more recently been found to be caused by de novo loss-of-function variants in TCEAL1. So far, a single study reported six unrelated individuals with this monogenetic disorder, presenting with syndromic features including developmental delay especially affecting expressive speech, intellectual disability, autistic-like behaviors, hypotonia, gait abnormalities and mild facial dysmorphism, in addition to ocular, gastrointestinal, and immunologic abnormalities. Here we report on four previously undescribed individuals, including two adults, with de novo truncating variants in TCEAL1, identified through trio exome or genome sequencing, further delineating the phenotype of the TCEAL1-related disorder. Whereas overall we identify similar features compared to the original report, we also highlight features in our adult individuals including hyperphagia, obesity, and endocrine abnormalities including hyperinsulinemia, hyperandrogenemia, and polycystic ovarian syndrome. X chromosome inactivation and RNA-seq studies further provide functional insights in the molecular mechanisms. Together this report expands the phenotypic and molecular spectrum of the TCEAL1-related disorder which will be useful for counseling of newly identified individuals and their families.

The role of a multidisciplinary team in managing variants of uncertain clinical significance in prenatal genetic diagnosis.

BACKGROUND: Although in general prenatal exome sequencing only reports (likely) pathogenic variants, in some cases a variant of uncertain significance (VUS) is disclosed. The aims of this retrospective study were to evaluate the types of VUS that have been reported to prospective parents, possible reclassification and to design a standard flow chart to determine which types of VUS could be considered for reporting in prenatal settings. Furthermore, we investigated what the crucial elements are to facilitate rapid management of uncertain results in a prenatal setting. MATERIAL AND METHODS: We reviewed exome results from 451 pregnancies performed in 2019-2021. We analyzed which factors that were taken into account by the multidisciplinary team (MDT) contributed towards decision making on reporting VUS after prenatal exome sequencing. RESULTS: In 9/451 (2%) pregnancies tested with exome sequencing using a broad panel analysis a VUS was reported. After birth 3/9 VUS could be reclassified to likely pathogenic variants based on new clinical follow up data. We considered reporting VUS in genes: 1) matching the fetal phenotype, 2) associated with a severe disorder when a functional test is available or 3) possibly associated with a disorder where early post-partum diagnosis and treatment are crucial for a better prognosis. Two flowcharts were designed to guide first the laboratory specialist and then the MDT in decisions on reporting VUS. The crucial elements that enabled timely decisions on VUS disclosure were regular meetings, appropriate expertise, professional connections with other experts and psychological safety within the MDT. CONCLUSION: In this study three out of nine VUS could be re-classified as likely pathogenic after clinical follow-up. In order to protect pregnant couples from the burden of uncertain results, the genetic professionals have to take the responsibility to limit the reporting of VUS. This can be done not only by automated filtering of data, by following professional guidelines and by building standardized decision flows, but also by discussing individual cases considering personal situations and the involved disease and by sharing professional experience and responsibility in a multidisciplinary prenatal team setting.