On the reproductive capabilities of aneuploid human preimplantation embryos.

In IVF cycles, the application of aneuploidy testing at the blastocyst stage is quickly growing, and the latest reports estimate almost half of cycles in the US undergo preimplantation genetic testing for aneuploidies (PGT-A). Following PGT-A cycles, understanding the predictive value of an aneuploidy result is paramount for making informed decisions about the embryo's fate and utilization. Compelling evidence from non-selection trials strongly supports that embryos diagnosed with a uniform whole-chromosome aneuploidy very rarely result in the live birth of a healthy baby, while their transfer exposes women to significant risks of miscarriage and chromosomally abnormal pregnancy. On the other hand, embryos displaying low range mosaicism for whole chromosomes have shown reproductive capabilities somewhat equivalent to uniformly euploid embryos, and they have comparable clinical outcomes and gestational risks. Therefore, given their clearly distinct biological origin and clinical consequences, careful differentiation between uniform and mosaic aneuploidy is critical in both the clinical setting when counseling individuals and in the research setting when presenting aneuploidy studies in human embryology. Here, we focus on the evidence gathered so far on PGT-A diagnostic predictive values and reproductive outcomes observed across the broad spectrum of whole-chromosome aneuploidies detected at the blastocyst stage to obtain evidence-based conclusions on the clinical management of aneuploid embryos in the quickly growing PGT-A clinical setting.

Limits imposed by the experimental design of a large prospective non-inferiority study on PGT-A invalidate many of the conclusions.

The New England Journal of Medicine recently published a large study addressing the efficacy of preimplantation genetic testing for aneuploidy (PGT-A). The 14-centre randomized control non-inferiority trial used cumulative live birth rate (CLBR) as a clinical endpoint to examine the value of PGT-A and concluded that conventional IVF was not inferior to IVF with PGT-A. Unfortunately, the experimental design was highly flawed; and in fact, the data generated in the study do not support the major conclusions presented in the publication. The embryos in each patient's three-embryo pool, which were available for transfer, were selected solely by morphology. The investigators then randomized patients to either the PGT-A group or the control group. It is important to note that PGT-A screening in the study group was done only after the embryos were selected. PGT-A was not really used in a meaningful way, which would have been for the PGT-A results to help in selecting which embryos would be in the three-embryo group. Thus, the outcomes were wholly determined prior to the study intervention. The ultimate delivery rate for each group of three embryos was determined when they were selected by morphology. The randomization, which occurred after embryo selection, would assure equal distribution of those cohorts destined to deliver and those destined to fail to the two study groups, the PGT-A and control groups. Thus, there was no potential for PGT-A to enhance selection and thus no possible way to improve the cumulative outcomes. Since there was no possible way for the control group to be inferior, the experimental design precluded any chance of evaluating the primary endpoint of the study. The primary question of the study was never evaluated. Another serious flaw was that the study was initiated prior to knowing how to interpret the data provided in the PGT-A analytical result. Specifically, the design excluded mosaic embryos from transfer despite the literature demonstrating the significant reproductive potential for these embryos. When accounting for the lost deliveries induced by this non-evidence-based decision, the expected delivery rates in the two groups become virtually identical. That is an important issue because the data from the study actually demonstrate the safety of PGT-A without diminution in outcomes from the impact of trophectoderm biopsy or the discarding of competent embryos which had wrongfully been considered aneuploid. A final serious flaw in the experimental design and interpretation of the data surrounding the issue of the miscarriage rate. The investigators noted that the miscarriage rate was lower in the PGT-A group but stated that its impact was insufficient to alter the CLBR. Of course, by design, the CLBRs were limited to being equivalent. There was no potential for enhanced outcomes in the PGT-A group and thus no possibility that the lower risk of miscarriage in the PGT-A group would raise the CLBR. The benefit of a lower miscarriage rate is real and significant. Its relevance should not be diminished based on the lack of a change in the CLBR since that was never possible in this study. The investigators of the study concluded that the CLBR with conventional ART is equivalent to that with PGT-A, but a simple review of the experiment reassigns their genuine findings to those of a safety study. Significantly, the data in the study demonstrate that the intervention of PGT-A is safe. This study neither supports nor refutes the efficacy of clinical PGT-A.

USMG5 Ashkenazi Jewish founder mutation impairs mitochondrial complex V dimerization and ATP synthesis.

Leigh syndrome is a frequent, heterogeneous pediatric presentation of mitochondrial oxidative phosphorylation (OXPHOS) disease, manifesting with psychomotor retardation and necrotizing lesions in brain deep gray matter. OXPHOS occurs at the inner mitochondrial membrane through the integrated activity of five protein complexes, of which complex V (CV) functions in a dimeric form to directly generate adenosine triphosphate (ATP). Mutations in several different structural CV subunits cause Leigh syndrome; however, dimerization defects have not been associated with human disease. We report four Leigh syndrome subjects from three unrelated Ashkenazi Jewish families harboring a homozygous splice-site mutation (c.87 + 1G>C) in a novel CV subunit disease gene, USMG5. The Ashkenazi population allele frequency is 0.57%. This mutation produces two USMG5 transcripts, wild-type and lacking exon 3. Fibroblasts from two Leigh syndrome probands had reduced wild-type USMG5 mRNA expression and undetectable protein. The mutation did not alter monomeric CV expression, but reduced both CV dimer expression and ATP synthesis rate. Rescue with wild-type USMG5 cDNA in proband fibroblasts restored USMG5 protein, increased CV dimerization and enhanced ATP production rate. These data demonstrate that a recurrent USMG5 splice-site founder mutation in the Ashkenazi Jewish population causes autosomal recessive Leigh syndrome by reduction of CV dimerization and ATP synthesis.

Mutations in TMEM260 Cause a Pediatric Neurodevelopmental, Cardiac, and Renal Syndrome.

Despite the accelerated discovery of genes associated with syndromic traits, the majority of families affected by such conditions remain undiagnosed. Here, we employed whole-exome sequencing in two unrelated consanguineous kindreds with central nervous system (CNS), cardiac, renal, and digit abnormalities. We identified homozygous truncating mutations in TMEM260, a locus predicted to encode numerous splice isoforms. Systematic expression analyses across tissues and developmental stages validated two such isoforms, which differ in the utilization of an internal exon. The mutations in both families map uniquely to the long isoform, raising the possibility of an isoform-specific disorder. Consistent with this notion, RT-PCR of lymphocyte cell lines from one of the kindreds showed reduced levels of only the long isoform, which could be ameliorated by emetine, suggesting that the mutation induces nonsense-mediated decay. Subsequent in vivo testing supported this hypothesis. First, either transient suppression or CRISPR/Cas9 genome editing of zebrafish tmem260 recapitulated key neurological phenotypes. Second, co-injection of morphants with the long human TMEM260 mRNA rescued CNS pathology, whereas the short isoform was significantly less efficient. Finally, immunocytochemical and biochemical studies showed preferential enrichment of the long TMEM260 isoform to the plasma membrane. Together, our data suggest that there is overall reduced, but not ablated, functionality of TMEM260 and that attenuation of the membrane-associated functions of this protein is a principal driver of pathology. These observations contribute to an appreciation of the roles of splice isoforms in genetic disorders and suggest that dissection of the functions of these transcripts will most likely inform pathomechanism.

A homozygous deleterious CDK10 mutation in a patient with agenesis of corpus callosum, retinopathy, and deafness.

The primary cilium is a key organelle in numerous physiological and developmental processes. Genetic defects in the formation of this non-motile structure, in its maintenance and function, underlie a wide array of ciliopathies in human, including craniofacial, brain and heart malformations, and retinal and hearing defects. We used exome sequencing to study the molecular basis of disease in an 11-year-old female patient who suffered from growth retardation, global developmental delay with absent speech acquisition, agenesis of corpus callosum and paucity of white matter, sensorineural deafness, retinitis pigmentosa, vertebral anomalies, patent ductus arteriosus, and facial dysmorphism reminiscent of STAR syndrome, a suspected ciliopathy. A homozygous variant, c.870_871del, was identified in the CDK10 gene, predicted to cause a frameshift, p.Trp291Alafs*18, in the cyclin-dependent kinase 10 protein. CDK10 mRNAs were detected in patient cells and do not seem to undergo non-sense mediated decay. CDK10 is the binding partner of Cyclin M (CycM) and CDK10/CycM protein kinase regulates ciliogenesis and primary cilium elongation. Notably, CycM gene is mutated in patients with STAR syndrome. Following incubation, the patient cells appeared less elongated and more densely populated than the control cells suggesting that the CDK10 mutation affects the cytoskeleton. Upon starvation and staining with acetylated-tubulin, γ-tubulin, and Arl13b, the patient cells exhibited fewer and shorter cilia than control cells. These findings underscore the importance of CDK10 for the regulation of ciliogenesis. CDK10 defect is likely associated with a new form of ciliopathy phenotype; additional patients may further validate this association.

Utilizing ethnic-specific differences in minor allele frequency to recategorize reported pathogenic deafness variants.

Ethnic-specific differences in minor allele frequency impact variant categorization for genetic screening of nonsyndromic hearing loss (NSHL) and other genetic disorders. We sought to evaluate all previously reported pathogenic NSHL variants in the context of a large number of controls from ethnically distinct populations sequenced with orthogonal massively parallel sequencing methods. We used HGMD, ClinVar, and dbSNP to generate a comprehensive list of reported pathogenic NSHL variants and re-evaluated these variants in the context of 8,595 individuals from 12 populations and 6 ethnically distinct major human evolutionary phylogenetic groups from three sources (Exome Variant Server, 1000 Genomes project, and a control set of individuals created for this study, the OtoDB). Of the 2,197 reported pathogenic deafness variants, 325 (14.8%) were present in at least one of the 8,595 controls, indicating a minor allele frequency (MAF) > 0.00006. MAFs ranged as high as 0.72, a level incompatible with pathogenicity for a fully penetrant disease like NSHL. Based on these data, we established MAF thresholds of 0.005 for autosomal-recessive variants (excluding specific variants in GJB2) and 0.0005 for autosomal-dominant variants. Using these thresholds, we recategorized 93 (4.2%) of reported pathogenic variants as benign. Our data show that evaluation of reported pathogenic deafness variants using variant MAFs from multiple distinct ethnicities and sequenced by orthogonal methods provides a powerful filter for determining pathogenicity. The proposed MAF thresholds will facilitate clinical interpretation of variants identified in genetic testing for NSHL. All data are publicly available to facilitate interpretation of genetic variants causing deafness.

Deficiency of HTRA2/Omi is associated with infantile neurodegeneration and 3-methylglutaconic aciduria.

BACKGROUND: Cell survival critically depends on the integrity of mitochondria, which play a pivotal role during apoptosis. Extensive mitochondrial damage promotes release of pro-apoptotic factors from the intermembrane space of mitochondria. Released mitochondrial proteins include Smac/DIABLO and HTRA2/Omi, which inhibit the cytosolic E3 ubiquitin ligase XIAP and other inhibitors of apoptosis proteins. AIMS: Here we investigated the cause of extreme hypertonia at birth, alternating with hypotonia, with the subsequent appearance of extrapyramidal symptoms, lack of psychomotor development, microcephaly, intractable seizures and early death in four patients from two unrelated families. The patients showed lactic acidemia, 3-methylglutaconic aciduria, intermittent neutropenia, evolving brain atrophy and disturbed cristae structure in muscle mitochondria. METHODS AND RESULTS: Using whole-exome sequencing, we identified missplicing mutation and a 5 bp deletion in HTRA2, encoding HTRA2/Omi. This protein was completely absent from the patients' fibroblasts, whose growth was impaired and which were hypersensitive to apoptosis. Expression of HtrA2/Omi or of the proteolytically inactive HTRA2/Omi protein restored the cells' apoptotic resistance. However, cell growth was only restored by the proteolytically active protein. CONCLUSIONS: This is the first report of recessive deleterious mutations in HTRA2 in human. The clinical phenotype, the increased apoptotic susceptibility and the impaired cell growth recapitulate those observed in the Htra2 knockout mice and in mutant mice with proteolytically inactive HTRA2/Omi. Together, they underscore the importance of both chaperone and proteolytic activities of HTRA2/Omi for balanced apoptosis sensitivity and for brain development. Absence of HTRA2/Omi is associated with severe neurodegenerative disorder of infancy, abnormal mitochondria, 3-methylglutaconic aciduria and increased sensitivity to apoptosis.

Deficiency of the alkaline ceramidase ACER3 manifests in early childhood by progressive leukodystrophy.

BACKGROUND/AIMS: Leukodystrophies due to abnormal production of myelin cause extensive morbidity in early life; their genetic background is still largely unknown. We aimed at reaching a molecular diagnosis in Ashkenazi-Jewish patients who suffered from developmental regression at 6-13 months, leukodystrophy and peripheral neuropathy. METHODS: Exome analysis, determination of alkaline ceramidase activity catalysing the conversion of C18:1-ceramide to sphingosine and D-ribo-C12-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-phytoceramide to NBD-C12-fatty acid using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and thin layer chromatography, respectively, and sphingolipid analysis in patients' blood by LC-MS/MS. RESULTS: The patients were homozygous for p.E33G in the ACER3, which encodes a C18:1-alkaline ceramidase and C20:1-alkaline ceramidase. The mutation abolished ACER3 catalytic activity in the patients' cells and failed to restore alkaline ceramidase activity in yeast mutant strain. The levels of ACER3 substrates, C18:1-ceramides and dihydroceramides and C20:1-ceramides and dihydroceramides and other long-chain ceramides and dihydroceramides were markedly increased in the patients' plasma, along with that of complex sphingolipids, including monohexosylceramides and lactosylceramides. CONCLUSIONS: Homozygosity for the p.E33G mutation in the ACER3 gene results in inactivation of ACER3, leading to the accumulation of various sphingolipids in blood and probably in brain, likely accounting for this new form of childhood leukodystrophy.

Leukoencephalopathy and early death associated with an Ashkenazi-Jewish founder mutation in the Hikeshi gene.

BACKGROUND: Leukodystrophies are genetic white matter disorders affecting the formation or maintenance of myelin. Among the recently discovered genetic defects associated with leukodystrophies, several genes converge on a common mechanism involving protein transcription/translation and ER stress response. METHODS: The genetic basis of a novel congenital leukodystrophy, associated with early onset spastic paraparesis, acquired microcephaly and optic atrophy was studied in six patients from three unrelated Ashkenazi-Jewish families. To this end we used homozygosity mapping, exome analysis, western blot (Hikeshi, HSF1-pS326 and b-actin) in patient fibroblasts, indirect immunofluorescence (HSP70 and HSF1) in patient fibroblasts undergoing heat shock stress, nuclear injection of plasmids expressing Hikeshi or EGFP in patient fibroblasts, in situ hybridization and Immunoblot analysis of Hikeshi in newborn and adult mouse brain. RESULTS: All the patients were homozygous for a missense mutation, p.Val54Leu, in C11ORF73 encoding HSP70 nuclear transporter protein, Hikeshi. The mutation segregated with the disease in the families and was carried by 1:200 Ashkenazi-Jewish individuals. The mutation was associated with undetectable level of Hikeshi in the patients' fibroblasts and with lack of nuclear HSP70 during heat shock stress, a phenomenon which was reversed upon the introduction of normal human Hikeshi to the patients cells. Hikeshi was found to be expressed in central white matter of mouse brain. CONCLUSIONS: These data underscore the importance of Hikeshi for HSP70 relocation into the nucleus. It is likely that in the absence of Hikeshi, HSP70 cannot attenuate the multiple heat shock induced nuclear phenotypes, leaving the cells unprotected during heat shock stress. We speculate that the sudden death of three of the six patients following a short febrile illness and the life-threatening myo-pericarditis in the fourth are the result of excess extra-nuclear HSP70 level which initiates cytokine release or provide target for natural killer cells. Alternatively, nuclear HSP70 might play an active role in stressed cells protection.

A mutation in the THG1L gene in a family with cerebellar ataxia and developmental delay.

Autosomal-recessive cerebellar atrophy is usually associated with inactivating mutations and early-onset presentation. The underlying molecular diagnosis suggests the involvement of neuronal survival pathways, but many mechanisms are still lacking and most patients elude genetic diagnosis. Using whole exome sequencing, we identified homozygous p.Val55Ala in the THG1L (tRNA-histidine guanylyltransferase 1 like) gene in three siblings who presented with cerebellar signs, developmental delay, dysarthria, and pyramidal signs and had cerebellar atrophy on brain MRI. THG1L protein was previously reported to participate in mitochondrial fusion via its interaction with MFN2. Abnormal mitochondrial fragmentation, including mitochondria accumulation around the nuclei and confinement of the mitochondrial network to the nuclear vicinity, was observed when patient fibroblasts were cultured in galactose containing medium. Culturing cells in galactose containing media promotes cellular respiration by oxidative phosphorylation and the action of the electron transport chain thus stimulating mitochondrial activity. The growth defect of the yeast thg1Δ strain was rescued by the expression of either yeast Thg1 or human THG1L; however, clear growth defect was observed following the expression of the human p.Val55Ala THG1L or the corresponding yeast mutant. A defect in the protein tRNAHis guanylyltransferase activity was excluded by the normal in vitro G-1 addition to either yeast tRNAHis or human mitochondrial tRNAHis in the presence of the THG1L mutation. We propose that homozygosity for the p.Val55Ala mutation in THG1L is the cause of the abnormal mitochondrial network in the patient fibroblasts, likely by interfering with THG1L activity towards MFN2. This may result in lack of mitochondria in the cerebellar Purkinje dendrites, with degeneration of Purkinje cell bodies and apoptosis of granule cells, as reported for MFN2 deficient mice.

Hypomyelination and developmental delay associated with VPS11 mutation in Ashkenazi-Jewish patients.

BACKGROUND: The genetic heterogeneity of developmental delay and cognitive impairment is vast. The endocytic network is essential for neural development and synaptic plasticity by regulating the sorting of numerous transmembrane proteins. Disruption of the pathway can lead to neuronal pathology. Endosomal biogenesis relies on two Rab proteins, Rab5 and Rab7, which bind to two hexameric tethering complexes, the endosomal class C core vacuole/endosome tethering complex (CORVET) and the late endosomal/lysosomal homotypic fusion and protein sorting complex (HOPS). Both complexes consist of four core proteins and differ by their specific Rab-binding proteins. OBJECTIVES: To identify the molecular basis of a neurological disease, which consists of global developmental stagnation at 3-8 months, increasing appendicular spasticity, truncal hypotonia and acquired microcephaly, with variable seizure disorder, accompanied by thin corpus callosum, paucity of white matter and delayed myelination in eight patients from four unrelated Ashkenazi-Jewish (AJ) families. METHODS: Exome analysis, homozygosity mapping and Mup1-GFP transport assay in mutant yeast. RESULTS: Homozygosity for a missense mutation, p.Cys846Gly, in one of the endosomal biogenesis core proteins, VPS11, was identified in all the patients. This was shown to be a founder mutation with a carrier frequency of 0.6% in the AJ population. The homologous yeast mutant had moderate impairment of fusion of the late endosome to the vacuole in Mup1-GFP transport assay. CONCLUSIONS: We speculate that in neuronal cells, impairment of fusion of the late endosome to the vacuole would attenuate the degradation of plasma membrane receptors, thereby underlying the progressive neuronal phenotype in our patients. The VPS11 p.Cys846Gly mutation should be added to the AJ carrier screening panel.

Mutations in COQ4, an essential component of coenzyme Q biosynthesis, cause lethal neonatal mitochondrial encephalomyopathy.

BACKGROUND: The identification of the molecular basis of mitochondrial disorders continues to be challenging and expensive. The increasing usage of next-generation sequencing is facilitating the discovery of the genetic aetiology of heterogeneous phenotypes associated with these conditions. Coenzyme Q(10) (CoQ(10)) is an essential cofactor for mitochondrial respiratory chain complexes and other biochemical pathways. Mutations in genes involved in CoQ(10) biosynthesis cause primary CoQ(10) deficiency syndromes that can be treated with oral supplementation of ubiquinone. METHODS: We used whole exome sequencing to evaluate six probands from four unrelated families with clinical findings suggestive of a mitochondrial disorder. Clinical data were obtained by chart review, parental interviews, direct patient assessment and biochemical and pathological evaluation. RESULTS: We identified five recessive missense mutations in COQ4 segregating with disease in all four families. One mutation was found in a homozygous state in two unrelated Ashkenazi Jewish probands. All patients were female, and presented on the first day of life, and died in the neonatal period or early infancy. Clinical findings included hypotonia (6/6), encephalopathy with EEG abnormalities (4/4), neonatal seizures (3/6), cerebellar atrophy (4/5), cardiomyopathy (5/6) and lactic acidosis (4/6). Autopsy findings in two patients revealed neuron loss and reactive astrocytosis or cerebellar and brainstem hypoplasia and microdysgenesis. CONCLUSIONS: Mutations in COQ4 cause an autosomal recessive lethal neonatal mitochondrial encephalomyopathy associated with a founder mutation in the Ashkenazi Jewish population. The early mortality in our cohort suggests that COQ4 is an essential component of the multisubunit complex required for CoQ(10) biosynthesis.

Mutations in SLC1A4, encoding the brain serine transporter, are associated with developmental delay, microcephaly and hypomyelination.

BACKGROUND: L-serine plays an essential role in neuronal development and function. Although a non-essential amino acid, L-serine must be synthesised within the brain because of its poor permeability by the blood-brain barrier. Within the brain, its synthesis is confined to astrocytes, and its shuttle to neuronal cells is performed by a dedicated neutral amino acid transporter, ASCT1. METHODS AND RESULTS: Using exome analysis we identified the recessive mutations, p.E256K, p.L315fs, and p.R457W, in SLC1A4, the gene encoding ASCT1, in patients with developmental delay, microcephaly and hypomyelination; seizure disorder was variably present. When expressed in a heterologous system, the mutations did not affect the protein level at the plasma membrane but abolished or markedly reduced L-serine transport for p.R457W and p.E256K mutations, respectively. Interestingly, p.E256K mutation displayed a lower L-serine and alanine affinity but the same substrate selectivity as wild-type ASCT1. CONCLUSIONS: The clinical phenotype of ASCT1 deficiency is reminiscent of defects in L-serine biosynthesis. The data underscore that ASCT1 is essential in brain serine transport. The SLC1A4 p.E256K mutation has a carrier frequency of 0.7% in the Ashkenazi-Jewish population and should be added to the carrier screening panel in this community.

SNP array-based analyses of unbalanced embryos as a reference to distinguish between balanced translocation carrier and normal blastocysts.

PURPOSE: The purpose of the study is to validate a method that provides the opportunity to distinguish a balanced translocation carrier embryo from a truly normal embryo in parallel with comprehensive chromosome screening (CCS). METHODS: A series of translocation carrier couples that underwent IVF with single nucleotide polymorphism (SNP) array-based CCS on 148 embryos were included. Predictions of balanced or normal status of each embryo were made based upon embryonic SNP genotypes. In one case, microdeletion status was used to designate whether embryos were balanced or normal. In 10 additional cases, conventional karyotyping was performed on newborns in order to establish the true genetic status (balanced or normal) of the original transferred embryo. Finally, implantation potential of balanced or normal embryos was compared. RESULTS: Phasing SNPs using unbalanced embryos allowed accurate prediction of whether transferred embryos were balanced translocation carriers or truly normal in all cases completed to date (100 % concordance with conventional karyotyping of newborns). No difference in implantation potential of balanced or normal embryos was observed. CONCLUSIONS: This study demonstrates the validity of a CCS method capable of distinguishing normal from balanced translocation carrier embryos. The only prerequisite is the availability of parental DNA and an unbalanced IVF embryo, making the method applicable to the majority of carrier couples. In addition, the SNP array platform allows simultaneous CCS for aneuploidy with the same platform and from the same biopsy. Future work will involve prospective predictions to select normal embryos with subsequent karyotyping of the resulting newborns.

Genetic loss of SH2B3 in acute lymphoblastic leukemia.

The SH2B adaptor protein 3 (SH2B3) gene encodes a negative regulator of cytokine signaling with a critical role in the homeostasis of hematopoietic stem cells and lymphoid progenitors. Here, we report the identification of germline homozygous SH2B3 mutations in 2 siblings affected with developmental delay and autoimmunity, one in whom B-precursor acute lymphoblastic leukemia (ALL) developed. Mechanistically, loss of SH2B3 increases Janus kinase-signal transducer and activator of transcription signaling, promotes lymphoid cell proliferation, and accelerates leukemia development in a mouse model of NOTCH1-induced ALL. Moreover, extended mutation analysis showed homozygous somatic mutations in SH2B3 in 2 of 167 ALLs analyzed. Overall, these results demonstrate a Knudson tumor suppressor role for SH2B3 in the pathogenesis of ALL and highlight a possible link between genetic predisposition factors in the pathogenesis of autoimmunity and leukemogenesis.

Mutations in SLC35A3 cause autism spectrum disorder, epilepsy and arthrogryposis.

BACKGROUND: The heritability of autism spectrum disorder is currently estimated at 55%. Identification of the molecular basis of patients with syndromic autism extends our understanding of the pathogenesis of autism in general. The objective of this study was to find the gene mutated in eight patients from a large kindred, who suffered from autism spectrum disorder, arthrogryposis and epilepsy. METHODS AND RESULTS: By linkage analysis and exome sequencing, we identified deleterious mutations in SLC35A3 in these patients. SLC35A3 encodes the major Golgi uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter. In Golgi vesicles isolated from patient fibroblasts the transport of the respective nucleotide sugar was significantly reduced causing a massive decrease in the content of cell surface expressed highly branched N-glycans and a concomitant sharp increase of lower branched glycoforms. CONCLUSIONS: Spontaneous mutation in SLC35A3 has been discovered in cattle worldwide, recapitulating the human phenotype with arthrogryposis and additional skeletal defects known as Complex Vertebral Malformation syndrome. The skeletal anomalies in the mutant cattle and in our patients, and perhaps even the neurological symptoms are likely the consequence of the lack of high-branched N-glycans and the concomitant abundance of lower-branched glycoforms at the cell surface. This pattern has previously been associated with growth arrest and induction of differentiation. With this study, we add SLC35A3 to the gene list of autism spectrum disorders, and underscore the crucial importance of UDP-GlcNAc in the regulation of the N-glycan branching pathway in the Golgi apparatus.

Agenesis of corpus callosum and optic nerve hypoplasia due to mutations in SLC25A1 encoding the mitochondrial citrate transporter.

BACKGROUND: Agenesis of corpus callosum has been associated with several defects of the mitochondrial respiratory chain and the citric acid cycle. We now report the results of the biochemical and molecular studies of a patient with severe neurodevelopmental disease manifesting by agenesis of corpus callosum and optic nerve hypoplasia. METHODS AND RESULTS: A mitochondrial disease was suspected in this patient based on the prominent excretion of 2-hydroxyglutaric acid and Krebs cycle intermediates in urine and the finding of increased reactive oxygen species content and decreased mitochondrial membrane potential in her fibroblasts. Whole exome sequencing disclosed compound heterozygosity for two pathogenic variants in the SLC25A1 gene, encoding the mitochondrial citrate transporter. These variants, G130D and R282H, segregated in the family and were extremely rare in controls. The mutated residues were highly conserved throughout evolution and in silico modeling investigations indicated that the mutations would have a deleterious effect on protein function, affecting either substrate binding to the transporter or its translocation mechanism. These predictions were validated by the observation that a yeast strain harbouring the mutations at equivalent positions in the orthologous protein exhibited a growth defect under stress conditions and by the loss of activity of citrate transport by the mutated proteins reconstituted into liposomes. CONCLUSIONS: We report for the first time a patient with a mitochondrial citrate carrier deficiency. Our data support a role for citric acid cycle defects in agenesis of corpus callosum as already reported in patients with aconitase or fumarate hydratase deficiency.

Confirmation and pathogenicity of small copy number variations incidentally detected via a targeted next-generation sequencing-based PGT-A platform.

OBJECTIVE: To evaluate the technical accuracy, inheritance, and pathogenicity of small copy number variants (CNVs) detected by a targeted next-generation sequencing (NGS)-based PGT-A platform. DESIGN: Retrospective observational study performed between 2020-2022. SUBJECTS: 12,157 patients who underwent clinical PGT-A performed by targeted NGS for whole chromosome and large segmental aneuploidies. EXPOSURE: An incidental finding was reported when a CNV gain/loss of at least three consecutive amplicons appeared in at least two embryos from the same IVF cycle. MAIN OUTCOME MEASURES: The primary outcome measures were the specificity, incidence, inheritance, and pathogenicity of small CNVs detected by the PGT-A platform. Accuracy of the PGT-A platform CNV calls was assessed via concordance with the CNV calls (size and genomic location) on chromosomal microarray of the gamete provider(s). Parental origin of the CNV and pathogenicity classifications were also reported. RESULTS: In 75 of 12,157 unique PGT-A patients (0.62%;95%CI:0.5-0.8%), an incidental finding that met reporting criteria was identified. Chromosomal microarray follow-up was requested for all cases and results were received for one or both members of 65 reproductive couples. In all cases, one of the gamete providers was confirmed to have the CNV identified in the embryos (100.0%: N=65/65 95%CI:94.5-100). The identified CNV was of maternal origin in 34 cases (52.3%) and of paternal origin in 31 cases (47.7%). A significant correlation was identified between PGT-A-predicted CNV sizes and chromosomal microarray detected sizes (r=0.81) and genomic coordinates on parental DNA. Twenty-six (40%) of the CNVs were classified as benign/likely benign, 30 (46.2%) as a variant of uncertain significance (VUS), and 9 (13.8%) as pathogenic/likely pathogenic. CONCLUSION: Certain PGT-A platforms may enable the detection of inherited, small CNVs with extremely high specificity without prior knowledge of parental status. The majority of CNVs in this data set were confirmed to be benign/likely benign or a VUS; however, pathogenic/likely pathogenic CNVs associated with a broad range of phenotypic features may also be detected, although a reliable negative predictive value for small CNVs with current PGT-A technologies is unknown due to the many technical challenges.

Hereditary sensory autonomic neuropathy caused by a mutation in dystonin.

In 4 infants with a new lethal autonomic sensory neuropathy with clinical features similar to familial dysautonomia as well as contractures, we identified a deleterious mutation in the DST gene, using homozygosity mapping followed by exome sequencing. DST encodes dystonin, a cytoskeleton linker protein, and the mutation results in an unstable transcript. Interestingly, dystonin is significantly more abundant in cells of familial dysautonomia patients with IKBKAP (I-κ-B kinase complex-associated protein) mutation compared to fibroblasts of controls, suggesting that upregulation of dystonin is responsible for the milder course in familial dysautonomia. Homozygosity mapping followed by exome sequencing is a successful approach to identify mutated genes in rare monogenic disorders.

Two novel CCDC88C mutations confirm the role of DAPLE in autosomal recessive congenital hydrocephalus.

BACKGROUND: Human congenital non-syndromic hydrocephalus is a vastly heterogeneous condition. A subgroup of cases are not secondary to a specific cause (eg, a neural tube defect), and within this subgroup, autosomal recessive inheritance has been described. One homozygous mutation in the DAPLE (Dvl-associating protein with a high frequency of leucine residues) protein-encoding gene CCDC88C (coiled-coil domain containing 88C) has recently been reported in a single family. The role of this gene has not been validated in another family, and no other autosomal recessive gene has been reported. METHODS: We used homozygosity mapping and whole exome sequencing in two families with primary, non-syndromic congenital hydrocephalus from two different ethnic backgrounds. RESULTS: In each family, we identified a novel homozygous mutation of CCDC88C. One mutation produced a premature stop codon at position 312 of the protein, while the second mutation induced a frameshift in the last exon, producing a stop codon that truncated the extreme C-terminus of DAPLE, including the 2026-2028 Gly-Cys-Val motif known to bind the post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1) (PDZ) domain of Dishevelled. CONCLUSIONS: Our data validate CCDC88C as causing autosomal recessive, primary non-syndromic congenital hydrocephalus, suggesting this gene may be an important cause of congenital hydrocephalus, and underscore the important role of the C-terminal PDZ domain-binding motif in the DAPLE protein.