Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

Dyskeratosis congenita (DC) is an inherited bone-marrow-failure disorder characterized by a triad of mucocutaneous features that include abnormal skin pigmentation, nail dystrophy, and oral leucoplakia. Despite the identification of several genetic variants that cause DC, a significant proportion of probands remain without a molecular diagnosis. In a cohort of eight independent DC-affected families, we have identified a remarkable series of heterozygous germline variants in the gene encoding thymidylate synthase (TYMS). Although the inheritance appeared to be autosomal recessive, one parent in each family had a wild-type TYMS coding sequence. Targeted genomic sequencing identified a specific haplotype and rare variants in the naturally occurring TYMS antisense regulator ENOSF1 (enolase super family 1) inherited from the other parent. Lymphoblastoid cells from affected probands have severe TYMS deficiency, altered cellular deoxyribonucleotide triphosphate pools, and hypersensitivity to the TYMS-specific inhibitor 5-fluorouracil. These defects in the nucleotide metabolism pathway resulted in genotoxic stress, defective transcription, and abnormal telomere maintenance. Gene-rescue studies in cells from affected probands revealed that post-transcriptional epistatic silencing of TYMS is occurring via elevated ENOSF1. These cell and molecular abnormalities generated by the combination of germline digenic variants at the TYMS-ENOSF1 locus represent a unique pathogenetic pathway for DC causation in these affected individuals, whereas the parents who are carriers of either of these variants in a singular fashion remain unaffected.

Narrowing the diagnostic gap: Genomes, episignatures, long-read sequencing, and health economic analyses in an exome-negative intellectual disability cohort.

PURPOSE: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively. METHODS: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID. RESULTS: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis. CONCLUSION: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost.

SUFU haploinsufficiency causes a recognisable neurodevelopmental phenotype at the mild end of the Joubert syndrome spectrum.

BACKGROUND: Joubert syndrome (JS) is a recessively inherited ciliopathy characterised by congenital ocular motor apraxia (COMA), developmental delay (DD), intellectual disability, ataxia, multiorgan involvement, and a unique cerebellar and brainstem malformation. Over 40 JS-associated genes are known with a diagnostic yield of 60%-75%.In 2018, we reported homozygous hypomorphic missense variants of the SUFU gene in two families with mild JS. Recently, heterozygous truncating SUFU variants were identified in families with dominantly inherited COMA, occasionally associated with mild DD and subtle cerebellar anomalies. METHODS: We reanalysed next generation sequencing (NGS) data in two cohorts comprising 1097 probands referred for genetic testing of JS genes. RESULTS: Heterozygous truncating and splice-site SUFU variants were detected in 22 patients from 17 families (1.5%) with strong male prevalence (86%), and in 8 asymptomatic parents. Patients presented with COMA, hypotonia, ataxia and mild DD, and only a third manifested intellectual disability of variable severity. Brain MRI showed consistent findings characterised by vermis hypoplasia, superior cerebellar dysplasia and subtle-to-mild abnormalities of the superior cerebellar peduncles. The same pattern was observed in two out of three tested asymptomatic parents. CONCLUSION: Heterozygous truncating or splice-site SUFU variants cause a novel neurodevelopmental syndrome encompassing COMA and mild JS, which likely represent overlapping entities. Variants can arise de novo or be inherited from a healthy parent, representing the first cause of JS with dominant inheritance and reduced penetrance. Awareness of this condition will increase the diagnostic yield of JS genetic testing, and allow appropriate counselling about prognosis, medical monitoring and recurrence risk.

A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein.

Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology.

De novo putative loss-of-function variants in TAF4 are associated with a neuro-developmental disorder.

TATA-binding protein associated factor 4 (TAF4) is a subunit of the Transcription Factor IID (TFIID) complex, a central player in transcription initiation. Other members of this multimeric complex have been implicated previously as monogenic disease genes in human developmental disorders. TAF4 has not been described to date as a monogenic disease gene. We here present a cohort of eight individuals, each carrying de novo putative loss-of-function (pLoF) variants in TAF4 and expressing phenotypes consistent with a neuro-developmental disorder (NDD). Common features include intellectual disability, abnormal behavior, and facial dysmorphisms. We propose TAF4 as a novel dominant disease gene for NDD, and coin this novel disorder "TAF4-related NDD" (T4NDD). We place T4NDD in the context of other disorders related to TFIID subunits, revealing shared features of T4NDD with other TAF-opathies.

Genome sequencing in families with congenital limb malformations.

The extensive clinical and genetic heterogeneity of congenital limb malformation calls for comprehensive genome-wide analysis of genetic variation. Genome sequencing (GS) has the potential to identify all genetic variants. Here we aim to determine the diagnostic potential of GS as a comprehensive one-test-for-all strategy in a cohort of undiagnosed patients with congenital limb malformations. We collected 69 cases (64 trios, 1 duo, 5 singletons) with congenital limb malformations with no molecular diagnosis after standard clinical genetic testing and performed genome sequencing. We also developed a framework to identify potential noncoding pathogenic variants. We identified likely pathogenic/disease-associated variants in 12 cases (17.4%) including four in known disease genes, and one repeat expansion in HOXD13. In three unrelated cases with ectrodactyly, we identified likely pathogenic variants in UBA2, establishing it as a novel disease gene. In addition, we found two complex structural variants (3%). We also identified likely causative variants in three novel high confidence candidate genes. We were not able to identify any noncoding variants. GS is a powerful strategy to identify all types of genomic variants associated with congenital limb malformation, including repeat expansions and complex structural variants missed by standard diagnostic approaches. In this cohort, no causative noncoding SNVs could be identified.

Pathogenic variants in CDH11 impair cell adhesion and cause Teebi hypertelorism syndrome.

Teebi hypertelorism syndrome (THS; OMIM 145420) is a rare craniofacial disorder characterized by hypertelorism, prominent forehead, short nose with broad or depressed nasal root. Some cases of THS have been attributed to SPECC1L variants. Homozygous variants in CDH11 truncating the transmembrane and intracellular domains have been implicated in Elsahy-Waters syndrome (EWS; OMIM 211380) with hypertelorism. We report THS due to CDH11 heterozygous missense variants on 19 subjects from 9 families. All affected residues in the extracellular region of Cadherin-11 (CHD11) are highly conserved across vertebrate species and classical cadherins. Six of the variants that cluster around the EC2-EC3 and EC3-EC4 linker regions are predicted to affect Ca2+ binding that is required for cadherin stability. Two of the additional variants [c.164G > C, p.(Trp55Ser) and c.418G > A, p.(Glu140Lys)] are also notable as they are predicted to directly affect trans-homodimer formation. Immunohistochemical study demonstrates that CDH11 is strongly expressed in human facial mesenchyme. Using multiple functional assays, we show that five variants from the EC1, EC2-EC3 linker, and EC3 regions significantly reduced the cell-substrate trans adhesion activity and one variant from EC3-EC4 linker results in changes in cell morphology, focal adhesion, and migration, suggesting dominant negative effect. Characteristic features in this cohort included depressed nasal root, cardiac and umbilical defects. These features distinguished this phenotype from that seen in SPECC1L-related hypertelorism syndrome and CDH11-related EWS. Our results demonstrate heterozygous variants in CDH11, which decrease cell-cell adhesion and increase cell migratory behavior, cause a form of THS, as termed CDH11-related THS.

The Role of ZEB2 in Human CD8 T Lymphocytes: Clinical and Cellular Immune Profiling in Mowat-Wilson Syndrome.

The Zeb2 gene encodes a transcription factor (ZEB2) that acts as an important immune mediator in mice, where it is expressed in early-activated effector CD8 T cells, and limits effector differentiation. Zeb2 homozygous knockout mice have deficits in CD8 T cells and NK cells. Mowat-Wilson syndrome (MWS) is a rare genetic disease resulting from heterozygous mutations in ZEB2 causing disease by haploinsufficiency. Whether ZEB2 exhibits similar expression patterns in human CD8 T cells is unknown, and MWS patients have not been comprehensively studied to identify changes in CD8 lymphocytes and NK cells, or manifestations of immunodeficiency. By using transcriptomic assessment, we demonstrated that ZEB2 is expressed in early-activated effector CD8 T cells of healthy human volunteers following vaccinia inoculation and found evidence of a role for TGFß-1/SMAD signaling in these cells. A broad immunological assessment of six genetically diagnosed MWS patients identified two patients with a history of recurrent sinopulmonary infections, one of whom had recurrent oral candidiasis, one with lymphopenia, two with thrombocytopenia and three with detectable anti-nuclear antibodies. Immunoglobulin levels, including functional antibody responses to protein and polysaccharide vaccination, were normal. The MWS patients had a significantly lower CD8 T cell subset as % of lymphocytes, compared to healthy controls (median 16.4% vs. 25%, p = 0.0048), and resulting increased CD4:CD8 ratio (2.6 vs. 1.8; p = 0.038). CD8 T cells responded normally to mitogen stimulation in vitro and memory CD8 T cells exhibited normal proportions of subsets with important tissue-specific homing markers and cytotoxic effector molecules. There was a trend towards a decrease in the CD8 T effector memory subset (3.3% vs. 5.9%; p = 0.19). NK cell subsets were normal. This is the first evidence that ZEB2 is expressed in early-activated human effector CD8 T cells, and that haploinsufficiency of ZEB2 in MWS patients had a slight effect on immune function, skewing T cells away from CD8 differentiation. To date there is insufficient evidence to support an immunodeficiency occurring in MWS patients.

The expanding LARS2 phenotypic spectrum: HLASA, Perrault syndrome with leukodystrophy, and mitochondrial myopathy.

LARS2 variants are associated with Perrault syndrome, characterized by premature ovarian failure and hearing loss, and with an infantile lethal multisystem disorder: Hydrops, lactic acidosis, sideroblastic anemia (HLASA) in one individual. Recently we reported LARS2 deafness with (ovario) leukodystrophy. Here we describe five patients with a range of phenotypes, in whom we identified biallelic LARS2 variants: three patients with a HLASA-like phenotype, an individual with Perrault syndrome whose affected siblings also had leukodystrophy, and an individual with a reversible mitochondrial myopathy, lactic acidosis, and developmental delay. Three HLASA cases from two unrelated families were identified. All were males with genital anomalies. Two survived multisystem disease in the neonatal period; both have developmental delay and hearing loss. A 55-year old male with deafness has not displayed neurological symptoms while his female siblings with Perrault syndrome developed leukodystrophy and died in their 30s. Analysis of muscle from a child with a reversible myopathy showed reduced LARS2 and mitochondrial complex I levels, and an unusual form of degeneration. Analysis of recombinant LARS2 variant proteins showed they had reduced aminoacylation efficiency, with HLASA-associated variants having the most severe effect. A broad phenotypic spectrum should be considered in association with LARS2 variants.

Parental experiences of ultrarapid genomic testing for their critically unwell infants and children.

PURPOSE: To explore parental experiences of ultrarapid genomic testing for their critically unwell infants and children. METHODS: Parents of critically unwell children who participated in a national ultrarapid genomic diagnosis program were surveyed >12 weeks after genomic results return. Surveys consisted of custom questions and validated scales, including the Decision Regret Scale and Genomics Outcome Scale. RESULTS: With 96 survey invitations sent, the response rate was 57% (n = 55). Most parents reported receiving enough information during pretest (n = 50, 94%) and post-test (n = 44, 83%) counseling. Perceptions varied regarding benefits of testing, however most parents reported no or mild decision regret (n = 45, 82%). The majority of parents (31/52, 60%) were extremely concerned about the condition recurring in future children, regardless of actual or perceived recurrence risk. Parents whose child received a diagnostic result reported higher empowerment. CONCLUSION: This study provides valuable insight into parental experiences of ultrarapid genomic testing in critically unwell children, including decision regret, empowerment, and post-test reproductive planning, to inform design and delivery of rapid diagnosis programs. The findings suggest considerations for pre- and post-test counseling that may influence parental experiences during the testing process and beyond, such as the importance of realistically conveying the likelihood for clinical and/or personal utility.

Revealing hidden genetic diagnoses in the ocular anterior segment disorders.

PURPOSE: Ocular anterior segment disorders (ASDs) are clinically and genetically heterogeneous, and genetic diagnosis often remains elusive. In this study, we demonstrate the value of a combined analysis protocol using phenotypic, genomic, and pedigree structure data to achieve a genetic conclusion. METHODS: We utilized a combination of chromosome microarray, exome sequencing, and genome sequencing with structural variant and trio analysis to investigate a cohort of 41 predominantly sporadic cases. RESULTS: We identified likely causative variants in 54% (22/41) of cases, including 51% (19/37) of sporadic cases and 75% (3/4) of cases initially referred as familial ASD. Two-thirds of sporadic cases were found to have heterozygous variants, which in most cases were de novo. Approximately one-third (7/22) of genetic diagnoses were found in rarely reported or recently identified ASD genes including PXDN, GJA8, COL4A1, ITPR1, CPAMD8, as well as the new phenotypic association of Axenfeld-Rieger anomaly with a homozygous ADAMTS17 variant. The remainder of the variants were in key ASD genes including FOXC1, PITX2, CYP1B1, FOXE3, and PAX6. CONCLUSIONS: We demonstrate the benefit of detailed phenotypic, genomic, variant, and segregation analysis to uncover some of the previously "hidden" heritable answers in several rarely reported and newly identified ocular ASD-related disease genes.

Activating variants in PDGFRB result in a spectrum of disorders responsive to imatinib monotherapy.

More than 50 individuals with activating variants in the receptor tyrosine kinase PDGFRB have been reported, separated based on clinical features into solitary myofibromas, infantile myofibromatosis, Penttinen syndrome with premature aging and osteopenia, Kosaki overgrowth syndrome, and fusiform aneurysms. Despite their descriptions as distinct clinical entities, review of previous reports demonstrates substantial phenotypic overlap. We present a case series of 12 patients with activating variants in PDGFRB and review of the literature. We describe five patients with PDGFRB activating variants whose clinical features overlap multiple diagnostic entities. Seven additional patients from a large family had variable expressivity and late-onset disease, including adult onset features and two individuals with sudden death. Three patients were treated with imatinib and had robust and rapid response, including the first two reported infants with multicentric myofibromas treated with imatinib monotherapy and one with a recurrent p.Val665Ala (Penttinen) variant. Along with previously reported individuals, our cohort suggests infants and young children had few abnormal features, while older individuals had multiple additional features, several of which appeared to worsen with advancing age. Our analysis supports a diagnostic entity of a spectrum disorders due to activating variants in PDGFRB. Differences in reported phenotypes can be dramatic and correlate with advancing age, genotype, and to mosaicism in some individuals.

From maternal tending to adolescent befriending: The adolescent transition of social support.

Attachment theory holds that parental relationships have lifelong effects on offspring social lives. The tend-and-befriend hypothesis posits that female friendships among humans evolved as part of a primate-wide coping mechanism to mediate stress by relying on social support. Here we bridge developmental and evolutionary frameworks to examine adolescent girls' perception of their reliance on female friendship for social support, how perceptions of parental relationships affect peer relationships, and the extent to which parent and peer relationships buffer depressive symptoms. We predict perceived maternal relationship quality will be positively associated with close female friendships, and maternal relationships, paternal relationships, and female friendship will buffer depressive symptoms. Participants were adolescent girls from a summer science camp (N = 95). Participants filled out demographic information, social network surveys, the Parent-Adolescent Communication Scale, and the Center for Epidemiology Depression Scale. Data was analyzed with Pearson's correlations, t tests, and path analysis. Adolescent girls with few female friends, compared with girls who had more than two very close female friends, experienced more depressive symptoms (t = 3.382, p = .001, D = 0.784). Adolescent girls with few female friends experienced more depressive symptoms compared to girls with two or more very close female friends (t = 3.382, p = .001, D = 0.784). Stronger maternal and paternal relationships were associated with having more female friends (maternal: t = -3.213, p = .003, D = 0.837; paternal: t = -2.432; p = .017). In the path analysis model, only maternal relationship quality significantly predicted female friendship category (ß = .33, CR = 2.770, p < .006). Furthermore, participants with two or more very close female friends and higher paternal relationship quality had significantly fewer depressive symptoms (friends; ß = -.19, CR = -2.112, p = .035; paternal: ß = -.33, CR = -3.220, p < .001), and older participants had more depressive symptoms (ß = .17, CR = -1.931, p = .036). These results provide additional support for the tend-and-befriend hypothesis, suggesting that maternal tending sets the stage for close female friendships.

Feasibility of Ultra-Rapid Exome Sequencing in Critically Ill Infants and Children With Suspected Monogenic Conditions in the Australian Public Health Care System.

Importance: Widespread adoption of rapid genomic testing in pediatric critical care requires robust clinical and laboratory pathways that provide equitable and consistent service across health care systems. Objective: To prospectively evaluate the performance of a multicenter network for ultra-rapid genomic diagnosis in a public health care system. Design, Setting, and Participants: Descriptive feasibility study of critically ill pediatric patients with suspected monogenic conditions treated at 12 Australian hospitals between March 2018 and February 2019, with data collected to May 2019. A formal implementation strategy emphasizing communication and feedback, standardized processes, coordination, distributed leadership, and collective learning was used to facilitate adoption. Exposures: Ultra-rapid exome sequencing. Main Outcomes and Measures: The primary outcome was time from sample receipt to ultra-rapid exome sequencing report. The secondary outcomes were the molecular diagnostic yield, the change in clinical management after the ultra-rapid exome sequencing report, the time from hospital admission to the laboratory report, and the proportion of laboratory reports returned prior to death or hospital discharge. Results: The study population included 108 patients with a median age of 28 days (range, 0 days to 17 years); 34% were female; and 57% were from neonatal intensive care units, 33% were from pediatric intensive care units, and 9% were from other hospital wards. The mean time from sample receipt to ultra-rapid exome sequencing report was 3.3 days (95% CI, 3.2-3.5 days) and the median time was 3 days (range, 2-7 days). The mean time from hospital admission to ultra-rapid exome sequencing report was 17.5 days (95% CI, 14.6-21.1 days) and 93 reports (86%) were issued prior to death or hospital discharge. A molecular diagnosis was established in 55 patients (51%). Eleven diagnoses (20%) resulted from using the following approaches to augment standard exome sequencing analysis: mitochondrial genome sequencing analysis, exome sequencing-based copy number analysis, use of international databases to identify novel gene-disease associations, and additional phenotyping and RNA analysis. In 42 of 55 patients (76%) with a molecular diagnosis and 6 of 53 patients (11%) without a molecular diagnosis, the ultra-rapid exome sequencing result was considered as having influenced clinical management. Targeted treatments were initiated in 12 patients (11%), treatment was redirected toward palliative care in 14 patients (13%), and surveillance for specific complications was initiated in 19 patients (18%). Conclusions and Relevance: This study suggests feasibility of ultra-rapid genomic testing in critically ill pediatric patients with suspected monogenic conditions in the Australian public health care system. However, further research is needed to understand the clinical value of such testing, and the generalizability of the findings to other health care settings.

Identification of clinically actionable variants from genome sequencing of families with congenital heart disease.

PURPOSE: Congenital heart disease (CHD) affects up to 1% of live births. However, a genetic diagnosis is not made in most cases. The purpose of this study was to assess the outcomes of genome sequencing (GS) of a heterogeneous cohort of CHD patients. METHODS: Ninety-seven families with probands born with CHD requiring surgical correction were recruited for genome sequencing. At minimum, a proband-parents trio was sequenced per family. GS data were analyzed via a two-tiered method: application of a high-confidence gene screen (hcCHD), and comprehensive analysis. Identified variants were assessed for pathogenicity using the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines. RESULTS: Clinically relevant genetic variants in known and emerging CHD genes were identified. The hcCHD screen identified a clinically actionable variant in 22% of families. Subsequent comprehensive analysis identified a clinically actionable variant in an additional 9% of families in genes with recent disease associations. Overall, this two-tiered approach provided a clinically relevant variant for 31% of families. CONCLUSIONS: Interrogating GS data using our two-tiered method allowed identification of variants with high clinical utility in a third of our heterogeneous cohort. However, association of emerging genes with CHD etiology, and development of novel technologies for variant assessment and interpretation, will increase diagnostic yield during future reassessment of our GS data.

Nemaline myopathy and distal arthrogryposis associated with an autosomal recessive TNNT3 splice variant.

A male neonate presented with severe weakness, hypotonia, contractures and congenital scoliosis. Skeletal muscle specimens showed marked atrophy and degeneration of fast fibers with striking nemaline rods and hypertrophy of slow fibers that were ultrastructurally normal. A neuromuscular gene panel identified a homozygous essential splice variant in TNNT3 (chr11:1956150G > A, NM_006757.3:c.681+1G > A). TNNT3 encodes skeletal troponin-Tfast and is associated with autosomal dominant distal arthrogryposis. TNNT3 has not previously been associated with nemaline myopathy (NM), a rare congenital myopathy linked to defects in proteins associated with thin filament structure and regulation. cDNA studies confirmed pathogenic consequences of the splice variant, eliciting exon-skipping and intron retention events leading to a frameshift. Western blot showed deficiency of troponin-Tfast protein with secondary loss of troponin-Ifast . We establish a homozygous splice variant in TNNT3 as the likely cause of severe congenital NM with distal arthrogryposis, characterized by specific involvement of Type-2 fibers and deficiency of troponin-Tfast .

Nesprins anchor kinesin-1 motors to the nucleus to drive nuclear distribution in muscle cells.

During skeletal muscle development, nuclei move dynamically through myotubes in a microtubule-dependent manner, driven by the microtubule motor protein kinesin-1. Loss of kinesin-1 leads to improperly positioned nuclei in culture and in vivo. Two models have been proposed to explain how kinesin-1 functions to move nuclei in myotubes. In the cargo model, kinesin-1 acts directly from the surface of the nucleus, whereas in an alternative model, kinesin-1 moves nuclei indirectly by sliding anti-parallel microtubules. Here, we test the hypothesis that an ensemble of Kif5B motors acts from the nuclear envelope to distribute nuclei throughout the length of syncytial myotubes. First, using an inducible dimerization system, we show that controlled recruitment of truncated, constitutively active kinesin-1 motors to the nuclear envelope is sufficient to prevent the nuclear aggregation resulting from depletion of endogenous kinesin-1. Second, we identify a conserved kinesin light chain (KLC)-binding motif in the nuclear envelope proteins nesprin-1 and nesprin-2, and show that recruitment of the motor complex to the nucleus via this LEWD motif is essential for nuclear distribution. Together, our findings demonstrate that the nucleus is a kinesin-1 cargo in myotubes and that nesprins function as nuclear cargo adaptors. The importance of achieving and maintaining proper nuclear position is not restricted to muscle fibers, suggesting that the nesprin-dependent recruitment of kinesin-1 to the nuclear envelope through the interaction of a conserved LEWD motif with kinesin light chain might be a general mechanism for cell-type-specific nuclear positioning during development.

Noncoding copy-number variations are associated with congenital limb malformation.

PurposeCopy-number variants (CNVs) are generally interpreted by linking the effects of gene dosage with phenotypes. The clinical interpretation of noncoding CNVs remains challenging. We investigated the percentage of disease-associated CNVs in patients with congenital limb malformations that affect noncoding cis-regulatory sequences versus genes sensitive to gene dosage effects.MethodsWe applied high-resolution copy-number analysis to 340 unrelated individuals with isolated limb malformation. To investigate novel candidate CNVs, we re-engineered human CNVs in mice using clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing.ResultsOf the individuals studied, 10% harbored CNVs segregating with the phenotype in the affected families. We identified 31 CNVs previously associated with congenital limb malformations and four novel candidate CNVs. Most of the disease-associated CNVs (57%) affected the noncoding cis-regulatory genome, while only 43% included a known disease gene and were likely to result from gene dosage effects. In transgenic mice harboring four novel candidate CNVs, we observed altered gene expression in all cases, indicating that the CNVs had a regulatory effect either by changing the enhancer dosage or altering the topological associating domain architecture of the genome.ConclusionOur findings suggest that CNVs affecting noncoding regulatory elements are a major cause of congenital limb malformations.

TM6SF2 rs58542926 impacts lipid processing in liver and small intestine.

The transmembrane 6 superfamily member 2 (TM6SF2) loss-of-function variant rs58542926 is a genetic risk factor for nonalcoholic fatty liver disease and progression to fibrosis but is paradoxically associated with lower levels of hepatically derived triglyceride-rich lipoproteins. TM6SF2 is expressed predominantly in liver and small intestine, sites for triglyceride-rich lipoprotein biogenesis and export. In light of this, we hypothesized that TM6SF2 may exhibit analogous effects on both liver and intestine lipid homeostasis. To test this, we genotyped rs58542926 in 983 bariatric surgery patients from the Geisinger Medical Center for Nutrition and Weight Management, Geisinger Health System, in Pennsylvania and from 3,556 study participants enrolled in the Amish Complex Disease Research Program. Although these two cohorts have different metabolic profiles, carriers in both cohorts had improved fasting lipid profiles. Importantly, following a high-fat challenge, carriers in the Amish Complex Disease Research Program cohort exhibited significantly lower postprandial serum triglycerides, suggestive of a role for TM6SF2 in the small intestine. To gain further insight into this putative role, effects of TM6SF2 deficiency were studied in a zebrafish model and in cultured human Caco-2 enterocytes. In both systems TM6SF2 deficiency resulted in defects in small intestine metabolism in response to dietary lipids, including significantly increased lipid accumulation, decreased lipid clearance, and increased endoplasmic reticulum stress. CONCLUSIONS: These data strongly support a role of TM6SF2 in the regulation of postprandial lipemia, potentially through a similar function for TM6SF2 in the lipidation and/or export of both hepatically and intestinally derived triglyceride-rich lipoproteins. (Hepatology 2017;65:1526-1542).