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Background: Somatic and germline genetic alterations are significant drivers of cancer. Increasing integration of new technologies which profile these alterations requires timely, equitable and high-quality genetic counselling to facilitate accurate diagnoses and informed decision-making by patients and their families in preventive and clinical settings. This article aims to provide an overview of genetic counselling legislation and practice across European Union (EU) Member States to serve as a foundation for future European recommendations and action., Methods: National legislative databases of all 27 Member States were searched using terms relevant to genetic counselling, translated as appropriate. Interviews with relevant experts from each Member State were conducted to validate legislative search results and provide detailed insights into genetic counselling practice in each country., Results: Genetic counselling is included in national legislative documents of 22 of 27 Member States, with substantial variation in legal mechanisms and prescribed details (i.e. the 'who, what, when and where' of counselling). Practice is similarly varied. Workforce capacity (25 of 27 Member States) and genetic literacy (all Member States) were common reported barriers. Recognition and/or better integration of genetic counsellors and updated legislation and were most commonly noted as the 'most important change' which would improve practice., Conclusions: This review highlights substantial variability in genetic counselling across EU Member States, as well as common barriers notwithstanding this variation. Future recommendations and action should focus on addressing literacy and capacity challenges through legislative, regulatory and/or strategic approaches at EU, national, regional and/or local levels., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Public Health Association.)

The PTCD3 gene product (protein PTCD3 or MRPS39) forms the entry channel of the mitochondrial small ribosomal subunit and binds to single-stranded mRNA. Here, we expand on the clinical manifestations of PTCD3 pathogenic variants by describing an early-onset patient with Leigh-like syndrome and two patients with milder form of disease, with combined oxidative phosphorylation deficiency. A 34-year-old male and his 33-year-old sister both have horizontal nystagmus, pronounced rough tremor, truncal ataxia, dysmetria, spasticity and hyperreflexia. The basal respiration rate decreased significantly for the male patient and his mother ( p  < 0.0001) compared to the controls. The whole genome sequencing analysis revealed two heterozygous variants in the PTCD3 : c.1182T>A, p.(Tyr394Ter) and c.805C>T, p.(His269Tyr). Tyr394Ter variant ablates the C-terminal half of the protein, including a significant portion of the central fold. In silico modelling for the variant His269Tyr shows that the inclusion of the slightly larger tyrosine sidechain is well tolerated, with no significant change in either the position or the movement of the surrounding area. The third case is a 9-year-old boy, who has a global developmental delay, central hypotonia, hyperreflexia and abnormal MRI. PTCD3 pathogenic variant c.538+4A>G was identified by whole exome sequencing. To test the variant's effect on splicing, an RT-PCR experiment was performed, which revealed skipping of an out-of-frame exon 7., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Authors. JIMD Reports published by John Wiley & Sons Ltd on behalf of SSIEM.)

Variants of mitochondrial DNA (mtDNA) have been identified as risk factors for the development of Parkinson's disease (PD). However, the underlying pathogenetic mechanisms remain unclear. Cybrid models carrying various genotypes of mtDNA variants were tested for resistance to PD-simulating MPP + treatment. The most resistant line was selected for transcriptome profiling, revealing specific genes potentially influencing the resistant characteristic. We then conducted protein validation and molecular biological studies to validate the related pathways as the influential factor. Cybrids carrying the W3 mtDNA haplogroup demonstrated the most resistance to the MPP + treatment. In the transcriptome study, PPP1R15A was identified, while further study noted elevated expressions of the coding protein GADD34 across all cybrids. In the study of GADD34-related mitochondrial unfolding protein response (mtUPR), we found that canonical mtUPR, launched by the phosphate eIF2a, is involved in the resistant characteristic of specific mtDNA to MPP + treatment. Our study suggests that a lower expression of GADD34 in the late phase of mtUPR may prolong the mtUPR process, thereby benefitting protein homeostasis and facilitating cellular resistance to PD development. We herein demonstrate that GADD34 plays an important role in PD development and should be further investigated as a target for the development of therapies for PD.

Host-controlled intracellular accommodation of nitrogen-fixing bacteria is essential for the establishment of a functional Root Nodule Symbiosis (RNS). In many host plants, this occurs via transcellular tubular structures (infection threads - ITs) that extend across cell layers via polar tip-growth. Comparative phylogenomic studies have identified RPG ( RHIZOBIUM-DIRECTED POLAR GROWTH ) among the critical genetic determinants for bacterial infection. In Medicago truncatula , RPG is required for effective IT progression within root hairs but the cellular and molecular function of the encoded protein remains elusive. Here, we show that RPG resides in the protein complex formed by the core endosymbiotic components VAPYRIN (VPY) and LUMPY INFECTION (LIN) required for IT polar growth, co-localizes with both VPY and LIN in IT tip- and perinuclear-associated puncta of M. truncatula root hairs undergoing infection and is necessary for VPY recruitment into these structures. Fluorescence Lifetime Imaging Microscopy (FLIM) of phosphoinositide species during bacterial infection revealed that functional RPG is required to sustain strong membrane polarization at the advancing tip of the IT. In addition, loss of RPG functionality alters the cytoskeleton-mediated connectivity between the IT tip and the nucleus and affects the polar secretion of the cell wall modifying enzyme NODULE PECTATE LYASE (NPL). Our results integrate RPG into a core host machinery required to support symbiont accommodation, suggesting that its occurrence in plant host genomes is essential to co-opt a multimeric protein module committed to endosymbiosis to sustain IT-mediated bacterial infection., Competing Interests: BL, CS, DI, MR, TV, MB, SE, CL, TO No competing interests declared, (© 2023, Lace et al.)

Mitochondria are involved in many vital functions in living cells, including the synthesis of ATP by oxidative phosphorylation (OXPHOS) and regulation of nuclear gene expression through retrograde signaling. Leigh syndrome is a heterogeneous neurological disorder resulting from an isolated complex I deficiency that causes damage to mitochondrial energy production. The pathogenic mitochondrial DNA (mtDNA) variant m.13513G>A has been associated with Leigh syndrome. The present study investigated the effects of this mtDNA variant on the OXPHOS system and cell retrograde signaling. Transmitochondrial cytoplasmic hybrid (cybrid) cell lines harboring 50% and 70% of the m.13513G>A variant were generated and tested along with wild-type (WT) cells. The functionality of the OXPHOS system was evaluated by spectrophotometric assessment of enzyme activity and high-resolution respirometry. Nuclear gene expression was investigated by RNA sequencing and droplet digital PCR. Increasing levels of heteroplasmy were associated with reduced OXPHOS system complex I, IV, and I + III activities, and high-resolution respirometry also showed a complex I defect. Profound changes in transcription levels of nuclear genes were observed in the cell lines harboring the pathogenic mtDNA variant, indicating the physiological processes associated with defective mitochondria.

HINT1 is an ubiquitous homodimeric purine phosphoramidase belonging to the histidine-triad superfamily. In neurons, HINT1 stabilizes the interaction of different receptors and regulates the effects of their signaling disturbances. Changes in HINT1 gene are associated with autosomal recessive axonal neuropathy with neuromyotonia. Aim of the study was detailed description of patients' phenotype with HINT1 homozygous NM&#95;005340.7: c.110G>C (p.Arg37Pro) variant. Seven homozygous and three compound heterozygous patients were recruited and evaluated using standardized tests for CMT patients, in four patients' nerve ultrasonography was performed. The median age of symptom onset was 10 years (range 1-20), with initial complaints being distal lower limb weakness with gait impairment, combined with muscle stiffness, more pronounced in the hands than in the legs and worsened by cold. Arm muscles became involved later, presenting with distal weakness and hypotrophy. Neuromyotonia was present in all reported patients and is thus a diagnostic hallmark. Electrophysiological studies demonstrated axonal polyneuropathy. Impaired mental performance was observed in six out of ten cases. In all patients with HINT1 neuropathy, ultrasound examination showed significantly reduced muscle volume as well as spontaneous fasciculations and fibrillations. The nerve cross-sectional areas of the median and ulnar nerves were closer to the lower limits of the normal values. None of the investigated nerves had structural changes. Our findings broaden the phenotype of HINT1 -neuropathy and have implications for diagnostics and ultrasonographic evaluation of HINT1 -neuropathy patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rozevska, Rots, Gailite, Linde, Mironovs, Timcenko, Linovs, Locmele, Micule, Lace and Kenina.)

In plants, the topological organization of membranes has mainly been attributed to the cell wall and the cytoskeleton. Additionally, few proteins, such as plant-specific remorins have been shown to function as protein and lipid organizers. Root nodule symbiosis requires continuous membrane re-arrangements, with bacteria being finally released from infection threads into membrane-confined symbiosomes. We found that mutations in the symbiosis-specific SYMREM1 gene result in highly disorganized perimicrobial membranes. AlphaFold modelling and biochemical analyses reveal that SYMREM1 oligomerizes into antiparallel dimers and may form a higher-order membrane scaffolding structure. This was experimentally confirmed when expressing this and other remorins in wall-less protoplasts is sufficient where they significantly alter and stabilize de novo membrane topologies ranging from membrane blebs to long membrane tubes with a central actin filament. Reciprocally, mechanically induced membrane indentations were equally stabilized by SYMREM1. Taken together we describe a plant-specific mechanism that allows the stabilization of large-scale membrane conformations independent of the cell wall., (© 2023. The Author(s).)

Background and Objectives: Genetic testing has become an integral part of health care, allowing the confirmation of thousands of hereditary diseases, including neuromuscular disorders (NMDs). The reported average prevalence of individual inherited NMDs is 3.7-4.99 per 10,000. This number varies greatly in the selected populations after applying population-wide studies. The aim of this study was to evaluate the effect of genetic analysis as the first-tier test in patients with NMD and to calculate the disease prevalence and allelic frequencies for reoccurring genetic variants., Methods: Patients with NMD from Latvia with molecular tests confirming their diagnosis in 2008-2020 were included in this retrospective study., Results: Diagnosis was confirmed in 153 unique cases of all persons tested. Next-generation sequencing resulted in a detection rate of 37%. Two of the most common childhood-onset NMDs in our population were spinal muscular atrophy and dystrophinopathies, with a birth prevalence of 1.01 per 10,000 newborns and 2.08 per 10,000 (male newborn population), respectively. The calculated point prevalence was 0.079 per 10,000 for facioscapulohumeral muscular dystrophy type 1, 0.078 per 10,000 for limb-girdle muscular dystrophy, 0.073 per 10,000 for nondystrophic congenital myotonia, 0.052 per 10,000 for spinobulbar muscular atrophy, and 0.047 per 10,000 for type 1 myotonic dystrophy., Discussion: DNA diagnostics is a successful approach. The carrier frequencies of the common CAPN3 , FKRP , SPG11 , and HINT1 gene variants as well as that of the SMN1 gene exon 7 deletion in the population of Latvia are comparable with data from Europe. The carrier frequency of the CLCN1 gene variant c.2680C>T p.(Arg894Ter) is 2.11%, and consequently, congenital myotonia is the most frequent NMD in our population., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Craniofacial morphogenesis is highly complex, as is the anatomical region involved. Errors during this process, resulting in orofacial clefts, occur in more than 400 genetic syndromes. Some cases of cleft lip and/or palate (CLP) are caused by mutations in single genes; however, complex interactions between genetic and environmental factors are considered to be responsible for the majority of non-syndromic CLP development. The aim of the current study was to identify genetic risk factors in patients with isolated cleft palate (CP) by whole genome sequencing. Patients with isolated CP ( n = 30) recruited from the Riga Cleft Lip and Palate Centre, Institute of Stomatology, Riga, were analyzed by whole genome sequencing. Pathogenic or likely pathogenic variants were discovered in genes associated with CP ( TBX22 , COL2A1 , FBN1 , PCGF2 , and KMT2D ) in five patients; hence, rare disease variants were identified in 17% of patients with non-syndromic isolated CP. Our results were relevant to routine genetic counselling practice and genetic testing recommendations. Based on our data, we propose that all newborns with orofacial clefts should be offered genetic testing, at least for a panel of known CLP genes. Only if the results are negative and there is no suggestive family history or additional clinical symptoms (which would support additional exome or genome-wide investigation), should multifactorial empiric recurrence risk prediction tools be applied for families., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lace, Pajusalu, Livcane, Grinfelde, Akota, Mauliņa, Barkāne, Stavusis and Inashkina.)

Legumes have maintained the ability to associate with rhizobia to sustain the nitrogen-fixing root nodule symbiosis (RNS). In Medicago truncatula, the Nod factor (NF)-dependent intracellular root colonization by Sinorhizobium meliloti initiates from young, growing root hairs. They form rhizobial traps by physically curling around the symbiont. 1 , 2 Although alterations in root hair morphology like branching and swelling have been observed in other plants in response to drug treatments 3 or genetic perturbations, 4-6 full root hair curling represents a rather specific invention in legumes. The entrapment of the symbiont completes with its full enclosure in a structure called the "infection chamber" (IC), 1 , 2 , 7 , 8 from which a tube-like membrane channel, the "infection thread" (IT), initiates. 1 , 2 , 9 All steps of rhizobium-induced root hair alterations are aided by a tip-localized cytosolic calcium gradient, 10 , 11 global actin re-arrangements, and dense subapical fine actin bundles that are required for the delivery of Golgi-derived vesicles to the root hair tip. 7 , 12-14 Altered actin dynamics during early responses to NFs or rhizobia have mostly been shown in mutants that are affected in the actin-related SCAR/WAVE complex. 15-18 Here, we identified a polarly localized SYMBIOTIC FORMIN 1 (SYFO1) to be required for NF-dependent alterations in membrane organization and symbiotic root hair responses. We demonstrate that SYFO1 mediates a continuum between the plasma membrane and the cell wall that is required for the onset of rhizobial infections., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nemaline myopathy is a rare disorder affecting the muscle sarcomere. Mutations in nebulin gene ( NEB ) are known to be responsible for about 50% of nemaline myopathy cases. Nebulin is a giant protein which is formed integrally with the sarcomeric thin filament. This complex gene is under extensive alternative splicing giving rise to multiple isoforms. In this study, we report a 6-year-old boy presenting with general muscular weaknesses. Identification of rod-shaped structures in the patient' biopsy raised doubt about the presence of a nemaline myopathy. Next-generation sequencing was used to identify a causative mutation for the patient syndrome. A homozygous deep intronic substitution was found in the intron 144 of the NEB . The variant was predicted by in silico tools to create a new donor splice site. Molecular analysis has shown that the mutation could alter splicing events of the nebulin gene leading to a significant decrease of isoforms level. This change in the expression level of nebulin could give rise to functional consequences in the sarcomere. These results are consistent with the phenotypes observed in the patient. Such a discovery of variants in this gene will allow a better understanding of the involvement of nebulin in neuromuscular diseases and help find new treatments for the nemaline myopathy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Laflamme, Lace, Thonta Setty, Rioux, Labrie, Droit, Chrestian and Rivest.)

Pyridoxine-dependent epilepsy (PDE) is a relatively rare subgroup of epileptic disorders. They generally present in infancy as an early onset epileptic encephalopathy or seizures, refractory to standard treatments, with rapid and variable responses to vitamin B6 treatment. Whole exome sequencing of three unrelated families identified homozygous pathogenic mutation c.370&#95;373del, p.Asp124fs in PLPBP gene in five persons. Haplotype analysis showed a single shared profile for the affected persons and their parents, leading to a hypothesis about founder effect of the mutation in Saguenay-Lac-St-Jean region of French Canadians. All affected probands also shared one single mitochondrial haplotype T2b3 and two rare variations in the mitochondrial genome m.801A>G and m.5166A>G suggesting that a single individual female introduced PLPBP mutation c.370&#95;373del, p.Asp124fs in Quebec. The mutation p.Asp124fs causes a severe disease phenotype with delayed myelination and cortical/subcortical brain atrophy. The most noteworthy radiological finding in this Quebec founder mutation is the presence of the temporal cysts that can be used as a marker of the disease. Also, both patients, who are alive, had a history of prenatal supplements taken by their mothers as antiemetic medication with high doses of pyridoxine. In the context of suspected PDE in patients with neonatal refractory seizures, treatment with pyridoxine and/or Pyridoxal-5-phophate has to be started immediately and continued until the results of genetic analysis received. Even with early appropriate treatment, neurological outcome of our patient is still poor., Competing Interests: The authors declare no potential conflict of interest., (© 2021 The Authors. JIMD Reports published by John Wiley & Sons Ltd on behalf of SSIEM.)

There has been one previous report of a cohort of patients with variants in Chromodomain Helicase DNA-binding 3 (CHD3), now recognized as Snijders Blok-Campeau syndrome. However, with only three previously-reported patients with variants outside the ATPase/helicase domain, it was unclear if variants outside of this domain caused a clinically similar phenotype. We have analyzed 24 new patients with CHD3 variants, including nine outside the ATPase/helicase domain. All patients were detected with unbiased molecular genetic methods. There is not a significant difference in the clinical or facial features of patients with variants in or outside this domain. These additional patients further expand the clinical and molecular data associated with CHD3 variants. Importantly we conclude that there is not a significant difference in the phenotypic features of patients with various molecular disruptions, including whole gene deletions and duplications, and missense variants outside the ATPase/helicase domain. This data will aid both clinical geneticists and molecular geneticists in the diagnosis of this emerging syndrome.

The initiation of intracellular host cell colonization by symbiotic rhizobia in Medicago truncatula requires repolarization of root hairs, including the rearrangement of cytoskeletal filaments. The molecular players governing microtubule (MT) reorganization during rhizobial infections remain to be discovered. Here, we identified M. truncatula DEVELOPMENTALLY REGULATED PLASMA MEMBRANE POLYPEPTIDE (DREPP), a member of the MT binding DREPP/PCaP protein family, and investigated its functions during rhizobial infections. We show that rhizobial colonization of drepp mutant roots as well as transgenic roots overexpressing DREPP is impaired. DREPP relocalizes into symbiosis-specific membrane nanodomains in a stimulus-dependent manner. This subcellular segregation coincides with DREPP-dependent MT fragmentation and a partial loss of the ability to reorganize the MT cytoskeleton in response to rhizobia, which might rely on an interaction between DREPP and the MT-organizing protein SPIRAL2. Taken together, our results reveal that establishment of symbiotic associations in M. truncatula requires DREPP in order to regulate MT reorganization during initial root hair responses to rhizobia., (© 2020 ASPB. All rights reserved.)

Background: Primary microcephaly is defined as reduced occipital-frontal circumference noticeable before 36 weeks of gestation. Large amount of insults might lead to microcephaly including infections, hypoxia and genetic mutations. More than 16 genes are described in autosomal recessive primary microcephaly. However, the cause of microcephaly remains unclear in many cases after extensive investigations and genetic screening., Case Presentation: Here, we described the case of a boy with primary microcephaly who presented to a neurology clinic with short stature, global development delay, dyskinetic movement, strabismus and dysmorphic features. We performed microcephaly investigations and genetic panels. Then, we performed whole-exome sequencing to identify any genetic cause. Microcephaly investigations and genetic panels were negative, but we found a new D317V homozygous mutation in TELOE-2 interacting protein 2 (TTI2) gene by whole-exome sequencing. TTI2 is implicated in DNA damage response and mutation in that gene was previously described in mental retardation, autosomal recessive 39., Conclusions: We described the first French Canadian case with primary microcephaly and global developmental delay secondary to a new D317V homozygous mutation in TTI2 gene. Our report also highlights the importance of TTI2 protein in brain development.

Background: HSD10 mitochondrial disease (HSD10MD), originally described as a deficiency of 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD), is a rare X-linked disorder of a moonlighting protein encoded by the HSD17B10. The diagnosis is usually first suspected on finding elevated isoleucine degradation metabolites in urine, reflecting decreased MHBD activity. However, it is now known that clinical disease pathogenesis reflects other independent functions of the HSD10 protein; particularly its essential role in mitochondrial transcript processing and tRNA maturation. The classical phenotype of HSD10MD in affected males is an infantile-onset progressive neurodegenerative disorder associated with severe mitochondrial dysfunction., Patients, Methods, and Results: In four unrelated families, we identified index patients with MHBD deficiency, which implied a diagnosis of HSD10MD. Each index patient was independently investigated because of neurological or developmental concerns. All had persistent elevations of urinary 2-methyl-3-hydroxybutyric acid and tiglylglycine. Analysis of HSD17B10 identified a single missense variant, c.364C>G, p.Leu122Val, in each case. This rare variant (1/183336 alleles in gnomAD) was previously reported in one Dutch patient and was described as pathogenic. The geographic origins of our families and results of haplotype analysis together provide evidence of a founder effect for this variant in Quebec. Notably, we identified an asymptomatic hemizygous adult male in one family, while a second independent genetic disorder contributed substantially to the clinical phenotypes observed in probands from two other families., Conclusion: The phenotype associated with p.Leu122Val in HSD17B10 currently appears to be attenuated and nonprogressive. This report widens the spectrum of phenotypic severity of HSD10MD and contributes to genotype-phenotype correlation. At present, we consider p.Leu122Val a "variant of uncertain significance." Nonetheless, careful follow-up of our patients remains advisable, to assess long-term clinical course and ensure appropriate management. It will also be important to identify other potential patients in our population and to characterize their phenotype., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Proteins anchored to the cell surface via glycosylphosphatidylinositol (GPI) play various key roles in the human body, particularly in development and neurogenesis. As such, many developmental disorders are caused by mutations in genes involved in the GPI biosynthesis and remodeling pathway. We describe ten unrelated families with bi-allelic mutations in PIGB, a gene that encodes phosphatidylinositol glycan class B, which transfers the third mannose to the GPI. Ten different PIGB variants were found in these individuals. Flow cytometric analysis of blood cells and fibroblasts from the affected individuals showed decreased cell surface presence of GPI-anchored proteins. Most of the affected individuals have global developmental and/or intellectual delay, all had seizures, two had polymicrogyria, and four had a peripheral neuropathy. Eight children passed away before four years old. Two of them had a clinical diagnosis of DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures), a condition that includes sensorineural deafness, shortened terminal phalanges with small finger and toenails, intellectual disability, and seizures; this condition overlaps with the severe phenotypes associated with inherited GPI deficiency. Most individuals tested showed elevated alkaline phosphatase, which is a characteristic of the inherited GPI deficiency but not DOORS syndrome. It is notable that two severely affected individuals showed 2-oxoglutaric aciduria, which can be seen in DOORS syndrome, suggesting that severe cases of inherited GPI deficiency and DOORS syndrome might share some molecular pathway disruptions., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Strigolactones (SLs) are plant hormones with various functions in development, responses to stress, and interactions with (micro)organisms in the rhizosphere, including with seeds of parasitic plants. Their perception for hormonal functions requires an α,β-hydrolase belonging to the D14 clade in higher plants; perception of host-produced SLs by parasitic seeds relies on similar but phylogenetically distinct proteins (D14-like). D14 and D14-like proteins are peculiar receptors, because they cleave SLs before undergoing a conformational change that elicits downstream events. Structure-activity relationship data show that the butenolide D-ring is crucial for bioactivity. We applied a bioisosteric approach to the structure of SLs by synthetizing analogues and mimics of natural SLs in which the D-ring was changed from a butenolide to a lactam and then evaluating their bioactivity. This was done by using a novel bioassay based on Arabidopsis transgenic lines expressing AtD14 fused to firefly luciferase, in parallel with the quantification of germination-inducing activity on parasitic seeds. The results obtained showed that the in planta bioassay is robust and quantitative, and thus can be confidently added to the SL-survey toolbox. The results also showed that modification of the butenolide ring into a lactam one significantly hampers the biological activity exhibited by SLs possessing a canonical lactonic D-ring.

Legumes have the almost unique ability to establish symbiotic associations with rhizobia and arbuscular mycorrhizal fungi. Forward and reverse genetics have identified a large number of genes that are required for either or both interactions. However, and in sharp contrast to natural soils, these interactions have been almost exclusively investigated under laboratory conditions by using separate inoculation systems, whereas both symbionts are simultaneously present in the field. Considering our recent understanding of the individual symbioses, the community is now promisingly positioned to co-inoculate plants with two or more microbes in order to understand mechanistically how legumes efficiently balance, regulate and potentially separate these symbioses and other endophytic microbes within the same root. Here, we discuss a number of key control layers that should be considered when assessing tri- or multipartite beneficial interactions and that may contribute to colonization patterns in legume roots.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency (mitochondrial HMG-CoA synthase deficiency or mHS deficiency, OMIM #605911) is an inborn error of metabolism that affects ketone body synthesis. Acute episodes include vomiting, lethargy, hepatomegaly, hypoglycemia and dicarboxylic aciduria. The diagnosis is difficult due to the relatively unspecific clinical and biochemical presentation, and fewer than 30 patients have been described. This work describes three new patients with mHS deficiency and two missense mutations c.334C>T (p.R112W) and c.430G>T (p.V144L) previously not reported. We developed a new method to express and measure the activity of the enzyme and in this work the study is extended to ten new missense variants including those of our patients. Enzymatic assays showed that three of the mutant proteins retained some but seven completely lacked activity. The identification of a patient homozygous for a mutation that retains 70% of enzyme activity opens the door to a new interpretation of the disease by demonstrating that a modest impairment of enzyme function can actually produce symptoms. This is also the first study employing molecular dynamics modelling of the enzyme mutations. We show that the correct maintenance of the dimerization surface is crucial for retaining the structure of the active center and therefore the activity of the enzyme., Competing Interests: The authors declare no conflict of interest.

Autosomal dominant North Carolina macular dystrophy (NCMD) is believed to represent a failure of macular development. The disorder has been linked to two loci, MCDR1 (chromosome 6q16) and MCDR3 (chromosome 5p15-p13). Recently, non-coding variants upstream of PRDM13 (MCDR1) and a duplication including IRX1 (MCDR3) have been identified. However, the underlying disease-causing mechanism remains uncertain. Through a combination of sequencing studies on eighteen NCMD families, we report two novel overlapping duplications at the MCDR3 locus, in a gene desert downstream of IRX1 and upstream of ADAMTS16. One duplication of 43 kb was identified in nine families (with evidence for a shared ancestral haplotype), and another one of 45 kb was found in a single family. Three families carry the previously reported V2 variant (MCDR1), while five remain unsolved. The MCDR3 locus is thus refined to a shared region of 39 kb that contains DNAse hypersensitive sites active at a restricted time window during retinal development. Publicly available data confirmed expression of IRX1 and ADAMTS16 in human fetal retina, with IRX1 preferentially expressed in fetal macula. These findings represent a major advance in our understanding of the molecular genetics of NCMD and provide insights into the genetic pathways involved in human macular development.

Modern biology overlaps with chemistry in explaining the structure and function of all cellular processes at the molecular level. Plant hormone research is perfectly located at the interface between these two disciplines, taking advantage of synthetic and computational chemistry as a tool to decipher the complex biological mechanisms regulating the action of plant hormones. These small signaling molecules regulate a wide range of developmental processes, adapting plant growth to ever changing environmental conditions. The synthesis of small bioactive molecules mimicking the activity of endogenous hormones allows us to unveil many molecular features of their functioning, giving rise to a new field, plant chemical biology. In this framework, fluorescence labeling of plant hormones is emerging as a successful strategy to track the fate of these challenging molecules inside living organisms. Thanks to the increasing availability of new fluorescent probes as well as advanced and innovative imaging technologies, we are now in a position to investigate many of the dynamic mechanisms through which plant hormones exert their action. Such a deep and detailed comprehension is mandatory for the development of new green technologies for practical applications. In this review, we summarize the results obtained so far concerning the fluorescent labeling of plant hormones, highlighting the basic steps leading to the design and synthesis of these compelling molecular tools and their applications., (Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.)

Introduction: Childhood acute lymphoblastic leukaemia (ALL) is a complex disease caused by a combination of genetic susceptibility and environmental exposure. Previous genome-wide association studies have reported several single nucleotide polymorphisms (SNPs) associated with the incidence of ALL. Several variations in genes encoding enzymes involved in carcinogenesis are suggested as being associated with an increased risk of ALL development., Material and Methods: We enrolled 77 paediatric ALL patients and 122 healthy controls, and in addition parental DNA was also available for 45 probands. SNPs rs10821936 (ARID5B), rs4132601 (IKZF1), rs2239633 (CEBPE), rs3731217 (CDKN2A) and rs1800566 (NQO1) and the presence of GSTT1 and GSTM1 null variants were detected. For statistical analysis the hybrid method of two designs 'Haplin' was used as well as linkage disequilibrium for family-based association studies., Results: We identified the SNP rs10821936 in the ARID5B gene as being statistically significantly associated with childhood ALL, especially if the C allele is in a homozygous state, relative risk (RR) 4.65, 95% CI: 2.03-10.6, p = 0.0006. Statistically significant differences were not found in other SNPs. We found risk combinations including all five variations, the strongest association being found in a combination where all five genetic variants are in a homozygous state, CCTTTTTTCC, p = 0.032., Conclusions: The identified SNP rs10821936 could serve as a potential risk marker for childhood ALL development. Further studies in an independent population are needed for verification.

Background: Limb-girdle muscular dystrophies are characterized by predominant involvement of the shoulder and pelvic girdle and trunk muscle groups. Currently, there are 31 genes implicated in the different forms of limb-girdle muscular dystrophies, which exhibit similar phenotypes and clinical overlap; therefore, advanced molecular techniques are required to achieve differential diagnosis., Methods: We investigated 26 patients from Latvia and 34 patients from Lithuania with clinical symptoms of limb-girdle muscular dystrophies, along with 565 healthy unrelated controls from general and ethnic populations using our developed test kit based on the Illumina VeraCode GoldenGate genotyping platform, Ion AmpliSeq Inherited Disease Panel and direct sequencing of mutations in calpain 3 (CAPN3), anoctamin 5 (ANO5) and fukutin related protein (FKRP) genes., Results: Analysis revealed a homozygous CAPN3 c.550delA mutation in eight patients and three heterozygous variants in controls: dysferlin (DYSF) c.5028delG, CAPN3 c.2288A > G, and FKRP c.135C > T. Additionally, three mutations within FKRP gene were found: homozygous c.826C > A, and two compound - c.826C > A/c.404&#95;405insT and c.826C > A/c.204&#95;206delCTC mutations, and one mutation within CLCN1 gene - c.2680C > T p.Arg894Ter. ANO5 c.191dupA was not present., Conclusions: Genetic diagnosis was possible in 12 of 60 patients (20%). The allele frequency of CAPN3 gene mutation c.550delA in Latvia is 0.0016 and in Lithuania - 0.0029. The allele frequencies of CAPN3 gene mutation c.2288A > G and DYSF gene mutation c.4872delG are 0.003.

Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10(-5); ORallelic = 2.46 [95% CI 1.6-3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10(-9)). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO., (Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Variations of the nonrecombining Y-chromosomal region were investigated in 159 unrelated Baltic-speaking ethnic Latvians from four different geographic regions, using 28 biallelic markers and 12 short tandem repeats. Eleven different haplogroups (hgs) were detected in a regionally homogeneous Latvian population, among which N1c, R1a, and I1 cover more than 85% of its paternal lineages. When compared its closest geographic neighbors, the composition of the Latvian Y-chromosomal gene pool was found to be very similar to those of Lithuanians and Estonians. Despite the comparable frequency distribution of hg N1c in Latvians and Lithuanians with the Finno-Ugric-speaking populations from the Eastern coast of the Baltic Sea, the observed differences in allelic variances of N1c haplotypes between these two groups are in concordance with the previously stated hypothesis of different dispersal ways of this lineage in the region. More than a third of Latvian paternal lineages belong specifically to a recently defined R1a-M558 hg, indicating an influence from a common source within Eastern Slavic populations on the formation of the present-day Latvian Y-chromosome gene pool., (© 2015 John Wiley & Sons Ltd/University College London.)

Background: Surgical resection is viewed as the most effective way to ensure both locoregional control and long-term survival in esophageal cancer. Although minimally invasive esophagectomy has been widely accepted as an alternative to open surgery, the role of robotic assistance has yet to be elucidated. We report our institutional experience with robotic-assisted Ivor Lewis esophagectomy using real-time perfusion assessment and demonstrate this as a safe and technically feasible alternative to traditional open Ivor Lewis esophagectomy., Methods: A retrospective chart review of all patients undergoing robotic-assisted Ivor Lewis esophagectomy at a single institution from 2011 to 2014 was performed. Operative and postoperative outcomes were recorded., Results: Fifty-four patients underwent robotic-assisted Ivor Lewis esophagectomy during the study period. Indication for surgery was cancer in 49 patients, 38 of whom underwent neoadjuvant chemoradiation therapy. The average operative time was 6 hours 2 minutes, and the average blood loss was 74 mL. There was 1 postoperative mortality (1.9%). Three (5.5%) patients experienced an anastomotic leak. The average number of lymph nodes harvested in cancer patients was 16.2 (range, 3 to 35). The average length of stay was 12.9 days., Conclusions: Our study demonstrates that robotic-assisted Ivor Lewis esophagectomy using real-time perfusion assessment is a safe and technically feasible alternative to traditional open Ivor Lewis esophagectomy. It allows for R0 resection with adequate lymph node harvesting and a short hospital stay., (Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Patients with cutis laxa (CL) have wrinkled, sagging skin with decreased elasticity. Skin symptoms are associated with variable systemic involvement. The most common, genetically highly heterogeneous form of autosomal recessive CL, ARCL2, is frequently associated with variable metabolic and neurological symptoms. Progeroid symptoms, dysmorphic features, hypotonia and psychomotor retardation are highly overlapping in the early phase of these disorders. This makes the genetic diagnosis often challenging. In search for discriminatory symptoms, we prospectively evaluated clinical, neurologic, metabolic and genetic features in our patient cohort referred for suspected ARCL. From a cohort of 26 children, we confirmed mutations in genes associated with ARCL in 16 children (14 probands), including 12 novel mutations. Abnormal glycosylation and gyration abnormalities were mostly, but not always associated with ATP6V0A2 mutations. Epilepsy was most common in ATP6V0A2 defects. Corpus callosum dysgenesis was associated with PYCR1 and ALDH18A1 mutations. Dystonic posturing was discriminatory for PYCR1 and ALDH18A1 defects. Metabolic markers of mitochondrial dysfunction were found in one patient with PYCR1 mutations. So far unreported white matter abnormalities were found associated with GORAB and RIN2 mutations. We describe a large cohort of CL patients with neurologic involvement. Migration defects and corpus callosum hypoplasia were not always diagnostic for a specific genetic defect in CL. All patients with ATP6V0A2 defects had abnormal glycosylation. To conclude, central nervous system and metabolic abnormalities were discriminatory in this genetically heterogeneous group, although not always diagnostic for a certain genetic defect in CL.

Aim: To determine the frequencies of mutations that cause inherited monogenic liver disorders in patients with chronic hepatitis C., Methods: This study included 86 patients with chronic hepatitis C (55 men, 31 women; mean age at diagnosis, 38.36 ± 14.52 years) who had undergone antiviral therapy comprising pegylated interferon and ribavirin. Viral load, biochemical parameter changes, and liver biopsy morphological data were evaluated in all patients. The control group comprised 271 unrelated individuals representing the general population of Latvia for mutation frequency calculations. The most frequent mutations that cause inherited liver disorders [gene (mutation): ATP7B (H1069Q), HFE (C282Y, H63D), UGT1A1 (TA)7, and SERPINA1 (PiZ)] were detected by polymerase chain reaction (PCR), bidirectional PCR allele-specific amplification, restriction fragment length polymorphism analysis, and sequencing., Results: The viral genotype was detected in 80 of the 86 patients. Viral genotypes 1, 2, and 3 were present in 61 (76%), 7 (9%), and 12 (15%) patients, respectively. Among all 86 patients, 50 (58%) reached an early viral response and 70 (81%) reached a sustained viral response. All 16 patients who did not reach a sustained viral response had viral genotype 1. Case-control analysis revealed a statistically significant difference in only the H1069Q mutation between patients and controls (patients, 0.057; controls, 0.012; odds ratio, 5.514; 95%CI: 1.119-29.827, P = 0.022). However, the H1069Q mutation was not associated with antiviral treatment outcomes or biochemical indices. The (TA) 7 mutation of the UGT1A1 gene was associated with decreased ferritin levels (beta regression coefficient = -295.7, P = 0.0087)., Conclusion: Genetic mutations that cause inherited liver diseases in patients with hepatitis C should be studied in detail.

Background: Acute lymphoblastic leukemia (ALL) is a complex disease caused by interactions between hazardous exogenous or/and endogenous agents and many mild effect inherited susceptibility mutations. Some of them are known, but their functional roles still requireinvestigation. Age is a recognized risk factor; children with disease onset after the age of ten have worse prognosis, presumably also triggered by inherited factors., Materials and Methods: The MDR1 gene polymorphisms rs1045642, rs2032582 and MTHFR gene polymorphisms rs1801131 and rs1801133 were genotyped in 68 ALL patients in remission and 102 age and gender matched controls; parental DNA samples were also available for 42 probands., Results: No case control association was found between analyzed polymorphisms and a risk of childhood ALL development. Linkage disequilibrium was not observed in a family-based association study either. Only marginal association was observed between genetic marker rs2032582A and later disease onset (p=0.04)., Conclusions: Our data suggest that late age of ALL onset could be triggered by mild effect common alleles.

Originally identified as allelochemicals involved in plant-parasite interactions, more recently, Strigolactones (SLs) have been shown to play multiple key roles in the rhizosphere communication between plants and mycorrhizal fungi. Even more recent is the hormonal role ascribed to SLs which broadens the biological impact of these relatively simple molecules. In spite of the crucial and multifaceted biological role of SLs, there are no data on the receptor(s) which bind(s) such active molecules, neither in the producing plants nor in parasitic weeds or AM fungi. Information about the putative receptor of SLs can be gathered by means of structural, molecular, and genetic approaches. Our contribution on this topic is the design and synthesis of fluorescent labeled SL analogs to be used as probes for the detection in vivo of the receptor(s). Knowledge of the putative receptor structure will boost the research on analogs of the natural substrates as required for agricultural applications.

Limb-girdle muscular dystrophies (LGMDs) is a heterogeneous group of muscular dystrophies that mostly affect the pelvic and shoulder girdle muscle groups. We report here a case of neuromuscular disease associated with Dupuytren's contracture, which has never been described before as cosegregating with an autosomal dominant type of inheritance. Dupuytren's contracture is a common disease, especially in Northern Europe. Comorbid conditions associated with Dupuytren's contracture are repetitive trauma to the hands, diabetes, and seizures, but it has never before been associated with neuromuscular disease. We hypothesize that patients may harbor mutations in genes with functions related to neuromuscular disease and Dupuytren's contracture development.

Previous evidence from tooth agenesis studies suggested IRF6 and TGFA interact. Since tooth agenesis is commonly found in individuals with cleft lip/palate (CL/P), we used four large cohorts to evaluate if IRF6 and TGFA interaction contributes to CL/P. Markers within and flanking IRF6 and TGFA genes were tested using Taqman or SYBR green chemistries for case-control analyses in 1,000 Brazilian individuals. We looked for evidence of gene-gene interaction between IRF6 and TGFA by testing if markers associated with CL/P were overtransmitted together in the case-control Brazilian dataset and in the additional family datasets. Genotypes for an additional 142 case-parent trios from South America drawn from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), 154 cases from Latvia, and 8,717 individuals from several cohorts were available for replication of tests for interaction. Tgfa and Irf6 expression at critical stages during palatogenesis was analyzed in wild type and Irf6 knockout mice. Markers in and near IRF6 and TGFA were associated with CL/P in the Brazilian cohort (p<10(-6)). IRF6 was also associated with cleft palate (CP) with impaction of permanent teeth (p<10(-6)). Statistical evidence of interaction between IRF6 and TGFA was found in all data sets (p = 0.013 for Brazilians; p = 0.046 for ECLAMC; p = 10(-6) for Latvians, and p = 0.003 for the 8,717 individuals). Tgfa was not expressed in the palatal tissues of Irf6 knockout mice. IRF6 and TGFA contribute to subsets of CL/P with specific dental anomalies. Moreover, this potential IRF6-TGFA interaction may account for as much as 1% to 10% of CL/P cases. The Irf6-knockout model further supports the evidence of IRF6-TGFA interaction found in humans.

Background: Females with a total or partial deletion of the short arm of the X chromosome have variable features of Turner syndrome, but mental retardation (MR) rarely occurs. The haploinsufficiency of deleted genes that escape X-inactivation may explain the occurrence of MR and autism. Ornithine transcarbamylase (OTC) deficiency is the most common urea cycle disorder and is inherited in an X-linked semi-dominant trait, and the OTC gene maps to Xp21. Methods: We report on a girl with MR, epilepsy and biochemical changes characteristic of OTC deficiency but no identifiable point mutation in the OTC gene. Standard G-banding cytogenetic analysis, whole genome karyotyping, and X-inactivation studies were performed to determine the genetic etiology of the OTC deficiency in the patient. Results: Cytogenetic analysis and molecular karyotyping using SNP array revealed a deletion of the whole short arm of the X chromosome (Xp22.33-p11.1). Inactivation studies also revealed a completely skewed X-inactivation. Conclusion: Our patient presented with MR, epilepsy, and some evidence of reduced OTC activity, but performed genetic studies gave no explanation for this phenotype. We hope that this case report contributes to the understanding of the underlying genetic factors of the manifestation of X-linked disorders in female patients.