DDX3X-related neurodevelopmental disorder is one of the most common monogenic causes of intellectual disability in females, with currently >1000 females diagnosed worldwide. In contrast, reports on affected males with DDX3X variants are scarce. The limited knowledge on this X-linked disorder in males hinders the interpretation of hemizygous DDX3X variants in clinical practice. In this study, we present a new cohort of 19 affected males (from 17 unrelated families) with (possibly) disease-causing DDX3X variants, for whom we collected clinical and molecular data. Additionally, we reviewed the existing literature on 13 males with DDX3X variants. The phenotype in males is diverse, including intellectual disability, speech/language delays, behavioural challenges and structural brain abnormalities. The vast majority of males have missense variants, including two recurrent variants (p.(Arg351Gln) and p.(Arg488Cys)). No truncating variants have been reported, consistent with the presumed embryonic lethality of complete loss-of-function of DDX3X in males. In our novel cohort, 6/17 variants are de novo in the affected male and 3/17 variants are de novo in the mother. This study provides significant insights in the genetic and phenotypic spectrum of males with DDX3X variants, by presenting the data of a combined cohort (n = 32) of novel and published individuals. Our data show that variants in DDX3X can cause an X-linked neurodevelopmental disorder in males, with unaffected or mildly affected carrier females. These findings will aid the interpretation of hemizygous missense variants in DDX3X and can guide clinical management and counselling, in particular with regard to recurrence risks in the respective families.

BackgroundTrio exome sequencing can be used to investigate congenital abnormalities identified on pregnancy ultrasound, but its use in an Australian context has not been assessed.AimsAssess clinical outcomes and changes in management after expedited genomic testing in the prenatal period to guide the development of a model for widespread implementation.Materials and methodsForty‐three prospective referrals for whole exome sequencing, including 40 trios (parents and pregnancy), two singletons and one duo were assessed in a tertiary hospital setting with access to a state‐wide pathology laboratory. Diagnostic yield, turn‐around time (TAT), gestational age at reporting, pregnancy outcome, change in management and future pregnancy status were assessed for each family.ResultsA clinically significant genomic diagnosis was made in 15/43 pregnancies (35%), with an average TAT of 12 days. Gestational age at time of report ranged from 16 + 5 to 31 + 6 weeks (median 21 + 3 weeks). Molecular diagnoses included neuromuscular and skeletal disorders, RASopathies and a range of other rare Mendelian disorders. The majority of families actively used the results in pregnancy decision making as well as in management of future pregnancies.ConclusionsRapid second trimester prenatal genomic testing can be successfully delivered to investigate structural abnormalities in pregnancy, providing crucial guidance for current and future pregnancy management. The time‐sensitive nature of this testing requires close laboratory and clinical collaboration to ensure appropriate referral and result communication. We found the establishment of a prenatal coordinator role and dedicated reporting team to be important facilitators. We propose this as a model for genomic testing in other prenatal services.

Ionic calcium (Ca2+) is a key messenger in signal transduction and its mitochondrial uptake plays an important role in cell physiology. This uptake is mediated by the mitochondrial Ca2+ uniporter (MCU), which is regulated by EMRE (essential MCU regulator) encoded by the SMDT1 (single-pass membrane protein with aspartate rich tail 1) gene. This work presents the genetic, clinical and cellular characterization of two patients harbouring SMDT1 variants and presenting with muscle problems. Analysis of patient fibroblasts and complementation experiments demonstrated that these variants lead to absence of EMRE protein, induce MCU subcomplex formation and impair mitochondrial Ca2+ uptake. However, the activity of oxidative phosphorylation enzymes, mitochondrial morphology and membrane potential, as well as routine/ATP-linked respiration were not affected. We hypothesize that the muscle-related symptoms in the SMDT1 patients result from aberrant mitochondrial Ca2+ uptake.

Mitochondrial diseases require customized approaches for reproductive counseling, addressing differences in recurrence risks and reproductive options. The majority of mitochondrial diseases is caused by mutations in nuclear genes and segregate in a Mendelian way. Prenatal diagnosis (PND) or preimplantation genetic testing (PGT) are available to prevent the birth of another severely affected child. In at least 15%-25% of cases, mitochondrial diseases are caused by mitochondrial DNA (mtDNA) mutations, which can occur de novo (25%) or be maternally inherited. For de novo mtDNA mutations, the recurrence risk is low and PND can be offered for reassurance. For maternally inherited, heteroplasmic mtDNA mutations, the recurrence risk is often unpredictable, due to the mitochondrial bottleneck. PND for mtDNA mutations is technically possible, but often not applicable given limitations in predicting the phenotype. Another option for preventing the transmission of mtDNA diseases is PGT. Embryos with mutant load below the expression threshold are being transferred. Oocyte donation is another safe option to prevent the transmission of mtDNA disease to a future child for couples who reject PGT. Recently, mitochondrial replacement therapy (MRT) became available for clinical application as an alternative to prevent the transmission of heteroplasmic and homoplasmic mtDNA mutations.

Expanded carrier screening (ECS) entails a screening offer for multiple recessive disorders at the same time, and allows testing of individuals or couples regardless of ancestry or geographic origin. Children of consanguineous couples have a higher-than-average risk of manifesting autosomal recessive disorders. This study aims to contribute to the responsible implementation of ECS for consanguineous couples. Seven semi-structured interviews were conducted with consanguineous couples who had recently participated in Whole Exome Sequencing (WES)-based ECS at Maastricht University Medical Center (MUMC+), the Netherlands. The test offered at MUMC+ covers a large number of disease-related genes (~2000), including severe, relatively mild, early- and late-onset disorders. Respondents were interviewed about their views on, and experiences with participation in WES-based ECS. Overall, participation was experienced as worthwhile: it enabled respondents to make informed choices with regard to family planning as well as to take on the presumed parental responsibility to deliver their children as healthy as possible. Furthermore, our findings suggest that (1) true consent for having this test requires timely information about the possible implications of a positive test result for specific categories of findings, as well as about the success rates of the available reproductive options; (2) the clinical geneticist can play a pivotal part in informing participants as well as providing clear information about autosomal recessive inheritance; (3) more research is needed to explore what type of genetic risk information is considered 'meaningful' by participants and actually contributes to reproductive decision-making.

Purpose Consanguineous couples are at increased risk of being heterozygous for the same autosomal recessive (AR) disorder(s), with a 25% risk of affected offspring as a consequence. Until recently, comprehensive preconception carrier testing (PCT) for AR disorders was unavailable in routine diagnostics. Here we developed and implemented such a test in routine clinical care. Methods We performed exome sequencing (ES) for 100 consanguineous couples. For each couple, rare variants that could give rise to biallelic variants in offspring were selected. These variants were subsequently filtered against a gene panel consisting of ~2,000 genes associated with known AR disorders (OMIM-based). Remaining variants were classified according to American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines, after which only likely pathogenic and pathogenic (class IV/V) variants, present in both partners, were reported. Results In 28 of 100 tested consanguineous couples (28%), likely pathogenic and pathogenic variants not previously known in the couple or their family were reported conferring 25% risk of affected offspring. Conclusion ES-based PCT provides a powerful diagnostic tool to identify AR disease carrier status in consanguineous couples. Outcomes provided significant reproductive choices for a higher proportion of these couples than previous tests.

In a Dutch non-consanguineous patient having mitochondrial encephalomyopathy with complex I and complex IV deficiency, whole exome sequencing revealed two compound heterozygous variants in SLIRP. SLIRP gene encodes a stem-loop RNA-binding protein that regulates mitochondrial RNA expression and oxidative phosphorylation (OXPHOS). A frameshift and a deep-intronic splicing variant reduced the amount of functional wild-type SLIRP RNA to 5%. Consequently, in patient fibroblasts, MT-ND1, MT-ND6, and MT-CO1 expression was reduced. Lentiviral transduction of wild-type SLIRP cDNA in patient fibroblasts increased MT-ND1, MT-ND6, and MT-CO1 expression (2.5-7.2-fold), whereas mutant cDNAs did not. A fourfold decrease of citrate synthase versus total protein ratio in patient fibroblasts indicated that the resulting reduced mitochondrial mass caused the OXPHOS deficiency. Transduction with wild-type SLIRP cDNA led to a 2.4-fold increase of this ratio and partly restored OXPHOS activity. This confirmed causality of the SLIRP variants. In conclusion, we report SLIRP variants as a novel cause of mitochondrial encephalomyopathy with OXPHOS deficiency.

Objective We describe a third patient with brain small vessel disease 3 (BSVD3), being the first with a homozygous essential splice site variant in the COLGALT1 gene, with a more severe phenotype than the 2 children reported earlier. Methods Analysis of whole exome sequencing (WES) data of the child and parents was performed. We validated the missplicing of the homozygous variant using reverse transcription PCR and Sanger sequencing of the mRNA in a lymphocyte culture. Results The patient presented antenatally with porencephaly on ultrasound and MRI. Postnatally, he showed a severe developmental delay, refractory epilepsy, spastic quadriplegia, and a progressive hydrocephalus. WES revealed a homozygous canonical splice site variant NM_024656.3:c.625-2A>C. PCR and Sanger sequencing of the mRNA demonstrated that 2 cryptic splice sites are activated, causing a frameshift in the major transcript and in-frame deletion in a minor transcript. Conclusions We report a third patient with biallelic pathogenic variants in COLGALT1, confirming the role of this gene in autosomal recessive BSVD3.

Objective The purpose of this study was to explore the diagnostic yield and clinical utility of trio-based rapid whole exome sequencing (rWES) in pregnancies of fetuses with a wide range of congenital anomalies detected by ultrasound imaging. Methods In this observational study, we analyzed the first 54 cases referred to our laboratory for prenatal rWES to support clinical decision making, after the sonographic detection of fetal congenital anomalies. The most common identified congenital anomalies were skeletal dysplasia (n = 20), multiple major fetal congenital anomalies (n = 17) and intracerebral structural anomalies (n = 7). Results A conclusive diagnosis was identified in 18 of the 54 cases (33%). Pathogenic variants were detected most often in fetuses with skeletal dysplasia (n = 11) followed by fetuses with multiple major fetal congenital anomalies (n = 4) and intracerebral structural anomalies (n = 3). A survey, completed by the physicians for 37 of 54 cases, indicated that the rWES results impacted clinical decision making in 68% of cases. Conclusions These results suggest that rWES improves prenatal diagnosis of fetuses with congenital anomalies, and has an important impact on prenatal and peripartum parental and clinical decision making.

The original version of this Article contained an error in the spelling of the author Pleuntje J. van der Sluijs, which was incorrectly given as Eline (P. J.) van der Sluijs. This has now been corrected in both the PDF and HTML versions of the Article.

BACKGROUND Neuromuscular disorders (NMDs) are clinically and genetically heterogeneous. Accurate molecular genetic diagnosis can improve clinical management, provides appropriate genetic counseling and testing of relatives, and allows potential therapeutic trials. OBJECTIVE To establish the clinical utility of panel-based whole exome sequencing (WES) in NMDs in a population with children and adults with various neuromuscular symptoms. METHODS Clinical exome sequencing, followed by diagnostic interpretation of variants in genes associated with NMDs, was performed in a cohort of 396 patients suspected of having a genetic cause with a variable age of onset, neuromuscular phenotype, and inheritance pattern. Many had previously undergone targeted gene testing without results. RESULTS Disease-causing variants were identified in 75/396 patients (19%), with variants in the three COL6-genes (COL6A1, COL6A2 and COL6A3) as the most common cause of the identified muscle disorder, followed by variants in the RYR1 gene. Together, these four genes account for almost 25% of cases in whom a definite genetic cause was identified. Furthermore, likely pathogenic variants and/or variants of uncertain significance were identified in 95 of the patients (24%), in whom functional and/or segregation analysis should be used to confirm or reject the pathogenicity. In 18% of the cases with a disease-causing variant of which we received additional clinical information, we identified a genetic cause in genes of which the associated phenotypes did not match that of the patients. Hence, the advantage of panel-based WES is its unbiased approach. CONCLUSION Whole exome sequencing, followed by filtering for NMD genes, offers an unbiased approach for the genetic diagnostics of NMD patients. This approach could be used as a first-tier test in neuromuscular disorders with a high suspicion of a genetic cause. With uncertain results, functional testing and segregation analysis are needed to complete the evidence.

STUDY QUESTION Does germline selection (besides random genetic drift) play a role during the transmission of heteroplasmic pathogenic mitochondrial DNA (mtDNA) mutations in humans? SUMMARY ANSWER We conclude that inheritance of mtDNA is mutation-specific and governed by a combination of random genetic drift and negative and/or positive selection. WHAT IS KNOWN ALREADY mtDNA inherits maternally through a genetic bottleneck, but the underlying mechanisms are largely unknown. Although random genetic drift is recognized as an important mechanism, selection mechanisms are thought to play a role as well. STUDY DESIGN, SIZE, DURATION We determined the mtDNA mutation loads in 160 available oocytes, zygotes, and blastomeres of five carriers of the m.3243A>G mutation, one carrier of the m.8993T>G mutation, and one carrier of the m.14487T>C mutation. PARTICIPANTS/MATERIALS, SETTING, METHODS Mutation loads were determined in PGD samples using PCR assays and analysed mathematically to test for random sampling effects. In addition, a meta-analysis has been performed on mutation load transmission data in the literature to confirm the results of the PGD samples. MAIN RESULTS AND THE ROLE OF CHANCE By applying the Kimura distribution, which assumes random mechanisms, we found that mtDNA segregations patterns could be explained by variable bottleneck sizes among all our carriers (moment estimates ranging from 10 to 145). Marked differences in the bottleneck size would determine the probability that a carrier produces offspring with mutations markedly different than her own. We investigated whether bottleneck sizes might also be influenced by non-random mechanisms. We noted a consistent absence of high mutation loads in all our m.3243A>G carriers, indicating non-random events. To test this, we fitted a standard and a truncated Kimura distribution to the m.3243A>G segregation data. A Kimura distribution truncated at 76.5% heteroplasmy has a significantly better fit (P-value = 0.005) than the standard Kimura distribution. For the m.8993T>G mutation, we suspect a skewed mutation load distribution in the offspring. To test this hypothesis, we performed a meta-analysis on published blood mutation levels of offspring-mother (O-M) transmission for the m.3243A>G and m.8993T>G mutations. This analysis revealed some evidence that the O-M ratios for the m.8993T>G mutation are different from zero (P-value <0.001), while for the m.3243A>G mutation there was little evidence that the O-M ratios are non-zero. Lastly, for the m.14487T>G mutation, where the whole range of mutation loads was represented, we found no indications for selective events during its transmission. LARGE SCALE DATA All data are included in the Results section of this article. LIMITATIONS, REASON FOR CAUTION The availability of human material for the mutations is scarce, requiring additional samples to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS Our data show that non-random mechanisms are involved during mtDNA segregation. We aimed to provide the mechanisms underlying these selection events. One explanation for selection against high m.3243A>G mutation loads could be, as previously reported, a pronounced oxidative phosphorylation (OXPHOS) deficiency at high mutation loads, which prohibits oogenesis (e.g. progression through meiosis). No maximum mutation loads of the m.8993T>G mutation seem to exist, as the OXPHOS deficiency is less severe, even at levels close to 100%. In contrast, high mutation loads seem to be favoured, probably because they lead to an increased mitochondrial membrane potential (MMP), a hallmark on which healthy mitochondria are being selected. This hypothesis could provide a possible explanation for the skewed segregation pattern observed. Our findings are corroborated by the segregation pattern of the m.14487T>C mutation, which does not affect OXPHOS and MMP significantly, and its transmission is therefore predominantly determined by random genetic drift. Our conclusion is that mutation-specific selection mechanisms occur during mtDNA inheritance, which has implications for PGD and mitochondrial replacement therapy. STUDY FUNDING/COMPETING INTEREST(S) This work has been funded by GROW-School of Oncology and Developmental Biology. The authors declare no competing interests.

One of the most common mitochondrial DNA (mtDNA) mutations, the A to G transition at base pair 3243, has been linked to changes in the brain, in addition to commonly observed hearing problems, diabetes and myopathy. However, a detailed quantitative description of m.3243A> G patients' brains has not been provided so far. In this study, ultra-high field MRI at 7T and volume-and surface-based data analyses approaches were used to highlight morphology (i.e. atrophy)-, microstructure (i.e. myelin and iron concentration)-and metabolism (i.e. cerebral blood flow)-related differences between patients (N = 22) and healthy controls (N = 15). The use of quantitative MRI at 7T allowed us to detect subtle changes of biophysical processes in the brain with high accuracy and sensitivity, in addition to typically assessed lesions and atrophy. Furthermore, the effect of m. 3243A> G mutation load in blood and urine epithelial cells on these MRI measures was assessed within the patient population and revealed that blood levels were most indicative of the brain's state and disease severity, based on MRI as well as on neuropsychological data. Morphometry MRI data showed a wide-spread reduction of cortical, subcortical and cerebellar gray matter volume, in addition to significantly enlarged ventricles. Moreover, surface-based analyses revealed brain area-specific changes in cortical thickness (e.g. of the auditory cortex), and in T-1, T-2* and cerebral blood flow as a function of mutation load, which can be linked to typically m. 3243A> G-related clinical symptoms (e.g. hearing impairment). In addition, several regions linked to attentional control (e.g. middle frontal gyrus), the sensorimotor network (e.g. banks of central sulcus) and the default mode network (e.g. precuneus) were characterized by alterations in cortical thickness, T-1, T-2* and/or cerebral blood flow, which has not been described in previous MRI studies. Finally, several hypotheses, based either on vascular, metabolic or astroglial implications of the m. 3243A> G mutation, are discussed that potentially explain the underlying pathobiology. To conclude, this is the first 7T and also the largest MRI study on this patient population that provides macroscopic brain correlates of the m. 3243A> G mutation indicating potential MRI biomarkers of mitochondrial diseases and might guide future (longitudinal) studies to extensively track neuropathological and clinical changes.

Mitochondrial disorders, characterized by clinical symptoms and/or OXPHOS deficiencies, are caused by pathogenic variants in mitochondrial genes. However, pathogenic variants in some of these genes can lead to clinical manifestations which overlap with other neuromuscular diseases, which can be caused by pathogenic variants in non-mitochondrial genes as well. Mitochondrial pathogenic variants can be found in the mitochondrial DNA (mtDNA) or in any of the 1,500 nuclear genes with a mitochondrial function. We have performed a two-step next-generation sequencing approach in a cohort of 117 patients, mostly children, in whom a mitochondrial disease-cause could likely or possibly explain the phenotype. A total of 86 patients had a mitochondrial disorder, according to established clinical and biochemical criteria. The other 31 patients had neuromuscular symptoms, where in a minority a mitochondrial genetic cause is present, but a non-mitochondrial genetic cause is more likely. All patients were screened for pathogenic variants in the mtDNA and, if excluded, analyzed by whole exome sequencing (WES). Variants were filtered for being pathogenic and compatible with an autosomal or X-linked recessive mode of inheritance in families with multiple affected siblings and/or consanguineous parents. Non-consanguineous families with a single patient were additionally screened for autosomal and X-linked dominant mutations in a predefined gene-set. We identified causative pathogenic variants in the mtDNA in 20% of the patient-cohort, and in nuclear genes in 49%, implying an overall yield of 68%. We identified pathogenic variants in mitochondrial and non-mitochondrial genes in both groups with, obviously, a higher number of mitochondrial genes affected in mitochondrial disease patients. Furthermore, we show that 31% of the disease-causing genes in the mitochondrial patient group were not included in the MitoCarta database, and therefore would have been missed with MitoCarta based gene-panels. We conclude that WES is preferable to panel-based approaches for both groups of patients, as the mitochondrial gene-list is not complete and mitochondrial symptoms can be secondary. Also, clinically and genetically heterogeneous disorders would require sequential use of multiple different gene panels. We conclude that WES is a comprehensive and unbiased approach to establish a genetic diagnosis in these patients, able to resolve multi-genic disease-causes.

This study aims to identify gene defects in pediatric cardiomyopathy and early-onset brain disease with oxidative phosphorylation (OXPHOS) deficiencies. We applied whole-exome sequencing in three patients with pediatric cardiomyopathy and early-onset brain disease with OXPHOS deficiencies. The brain pathology was studied by MRI analysis. In consanguineous patient 1, we identified a homozygous intronic variant (c.850-3A > G) in the QRSL1 gene, which was predicted to cause abnormal splicing. The variant segregated with the disease and affected the protein function, which was confirmed by complementation studies, restoring OXPHOS function only with wild-type QRSL1. Patient 2 was compound heterozygous for two novel affected and disease-causing variants (c.[253G > A];[938G > A]) in the MTO1 gene. In patient 3, we detected one unknown affected and disease-causing variants (c.2872C > T) and one known disease-causing variant (c.1774C > T) in the AARS2 gene. The c.1774C > T variant was present in the paternal copy of the AARS2 gene, the c.2872C > T in the maternal copy. All genes were involved in translation of mtDNA-encoded proteins. Defects in mtDNA-encoded protein translation lead to severe pediatric cardiomyopathy and brain disease with OXPHOS abnormalities. This suggests that the heart and brain are particularly sensitive to defects in mitochondrial protein synthesis during late embryonic or early postnatal development, probably due to the massive mitochondrial biogenesis occurring at that stage. If both the heart and brain are involved, the prognosis is poor with a likely fatal outcome at young age.

Whole-exome sequencing identified multiple genetic causes in 2 infants with heterogeneous disease. Three gene defects in the first patient explained all symptoms, but manifestations were overlapping (blended phenotype). Two gene defects in the second patient explained nonoverlapping symptoms (composite phenotype). Whole-exome sequencing rapidly and comprehensively resolves heterogeneous genetic disease.

In a 51-year-old patient of consanguineous parents with a severe neuromuscular phenotype of early-onset ataxia, myoclonia, dysarthria, muscle weakness and exercise intolerance, exome sequencing revealed a novel homozygous variant (c.-264_31delinsCTCACAAATGCTCA) in the mitochondrial FAD-transporter gene SLC25A32. Flavin adenine dinucleotide (FAD) is an essential co-factor for many mitochondrial enzymes and impaired mitochondrial FAD-transport was supported by a reduced oxidative phosphorylation complex II activity in the patient's muscle, decreased ATP production in fibroblasts, and a deficiency of mitochondrial FAD-dependent enzymes. Clinically, the patient showed improvement upon riboflavin treatment, which is a precursor of FAD. Our results confirm the recently reported case of SLC25A32 as a cause of riboflavin-responsive disease. Our patient showed a more severe clinical phenotype compared with the reported patient, corresponding with the (most likely) complete absence of the SLC25A32-encoding MFT (Mitochondrial Folate Transporter) protein.