Next-generation cytogenetics: Comprehensive assessment of 52 hematological malignancy genomes by optical genome mapping.

Somatic structural variants (SVs) are important drivers of cancer development and progression. In a diagnostic set-up, especially for hematological malignancies, the comprehensive analysis of all SVs in a given sample still requires a combination of cytogenetic techniques, including karyotyping, FISH, and CNV microarrays. We hypothesize that the combination of these classical approaches could be replaced by optical genome mapping (OGM). Samples from 52 individuals with a clinical diagnosis of a hematological malignancy, divided into simple (<5 aberrations, n = 36) and complex (≥5 aberrations, n = 16) cases, were processed for OGM, reaching on average: 283-fold genome coverage. OGM called a total of 918 high-confidence SVs per sample, of which, on average, 13 were rare and >100 kb. In addition, on average, 73 CNVs were called per sample, of which six were >5 Mb. For the 36 simple cases, all clinically reported aberrations were detected, including deletions, insertions, inversions, aneuploidies, and translocations. For the 16 complex cases, results were largely concordant between standard-of-care and OGM, but OGM often revealed higher complexity than previously recognized. Detailed technical comparison with standard-of-care tests showed high analytical validity of OGM, resulting in a sensitivity of 100% and a positive predictive value of >80%. Importantly, OGM resulted in a more complete assessment than any previous single test and most likely reported the most accurate underlying genomic architecture (e.g., for complex translocations, chromoanagenesis, and marker chromosomes). In conclusion, the excellent concordance of OGM with diagnostic standard assays demonstrates its potential to replace classical cytogenetic tests as well as to rapidly map novel leukemia drivers.

Optical genome mapping enables constitutional chromosomal aberration detection.

Chromosomal aberrations including structural variations (SVs) are a major cause of human genetic diseases. Their detection in clinical routine still relies on standard cytogenetics. Drawbacks of these tests are a very low resolution (karyotyping) and the inability to detect balanced SVs or indicate the genomic localization and orientation of duplicated segments or insertions (copy number variant [CNV] microarrays). Here, we investigated the ability of optical genome mapping (OGM) to detect known constitutional chromosomal aberrations. Ultra-high-molecular-weight DNA was isolated from 85 blood or cultured cells and processed via OGM. A de novo genome assembly was performed followed by structural variant and CNV calling and annotation, and results were compared to known aberrations from standard-of-care tests (karyotype, FISH, and/or CNV microarray). In total, we analyzed 99 chromosomal aberrations, including seven aneuploidies, 19 deletions, 20 duplications, 34 translocations, six inversions, two insertions, six isochromosomes, one ring chromosome, and four complex rearrangements. Several of these variants encompass complex regions of the human genome involved in repeat-mediated microdeletion/microduplication syndromes. High-resolution OGM reached 100% concordance compared to standard assays for all aberrations with non-centromeric breakpoints. This proof-of-principle study demonstrates the ability of OGM to detect nearly all types of chromosomal aberrations. We also suggest suited filtering strategies to prioritize clinically relevant aberrations and discuss future improvements. These results highlight the potential for OGM to provide a cost-effective and easy-to-use alternative that would allow comprehensive detection of chromosomal aberrations and structural variants, which could give rise to an era of "next-generation cytogenetics."

A 12q24.31 interstitial deletion in an adult male with MODY3: neuropsychiatric and neuropsychological characteristics.

A 39-year-old male patient with a disharmonic intelligence profile and juvenile diabetes mellitus is described. At 14 months of age, minor facial dysmorphisms were noticed. He had delayed motor development, obesity at early age, and a diagnosis of insulin-dependent diabetes at the age of 10 years. He successfully completed secondary education and has been engaged in unskilled work activities, living independently. Upon examination, no psychiatric symptoms were present and his neuropsychological profile showed normal, although disharmonic, intellectual capacities and suboptimal social cognition. Genome wide array analysis identified an interstitial 12q24.31 deletion of 1.67 Mb encompassing hepatocyte nuclear factor-1-alpha gene (HNF1A), supporting a diagnosis of maturity-onset diabetes of the young. Results are discussed in relation to the few identified or published overlapping deletions. This is the first patient with normal intelligence in whom the presence of subtle facial dysmorphisms were decisive for introducing genetic analysis that, in turn, disclosed a rare form of diabetes necessitating modifications in treatment regimen. Clinicians, including those involved in psychiatry, should be aware of the diagnostic and prognostic value of atypical physical features in patients with a long history of complicated glucose regulation.

Utilization of automated cilia analysis to characterize novel INPP5E variants in patients with non-syndromic retinitis pigmentosa.

INPP5E encodes inositol polyphosphate-5-phosphatase E, an enzyme involved in regulating the phosphatidylinositol (PIP) makeup of the primary cilium membrane. Pathogenic variants in INPP5E hence cause a variety of ciliopathies: genetic disorders caused by dysfunctional cilia. While the majority of these disorders are syndromic, such as the neuronal ciliopathy Joubert syndrome, in some cases patients will present with an isolated phenotype-most commonly non-syndromic retinitis pigmentosa (RP). Here, we report two novel variants in INPP5E identified in two patients with non-syndromic RP: patient 1 with compound heterozygous variants (c.1516C > T, p.(Q506*), and c.847G > A, p.(A283T)) and patient 2 with a homozygous variant (c.1073C > T, p.(P358L)). To determine whether these variants were causative for the phenotype in the patients, automated ciliary phenotyping of patient-derived dermal fibroblasts was performed for percent ciliation, cilium length, retrograde IFT trafficking, and INPP5E localization. In both patients, a decrease in ciliary length and loss of INPP5E localization in the primary cilia were seen. With these molecular findings, we can confirm functionally that the novel variants in INPP5E are causative for the RP phenotypes seen in both patients. Additionally, this study demonstrates the usefulness of utilizing ciliary phenotyping as an assistant in ciliopathy diagnosis and phenotyping.

Infant Milk Formula with Large, Milk Phospholipid-coated Lipid Droplets Enriched in Dairy Lipids Affects Body Mass Index Trajectories and Blood Pressure at School Age: Follow-up of a Randomized Controlled Trial.

BACKGROUND: Human milk comprises large fat globules enveloped by a native phospholipid membrane, whereas infant formulas contain small, protein-coated lipid droplets. Previous experimental studies indicated that mimicking the architecture of human milk lipid droplets in infant milk formula (IMF) alters lipid metabolism with lasting beneficial impact on later metabolic health. OBJECTIVES: To evaluate in a follow-up (FU) study of a randomized, controlled trial whether a Concept IMF with large, milk phospholipid-coated lipid droplets enriched with dairy lipids beneficially impacts long-term body mass index (BMI in kg/m2) trajectories and blood pressure at school age. METHODS: Fully formula-fed infants were randomly assigned to Concept IMF (n = 115) or Control IMF with conventional, small lipid droplets containing vegetable oils (n = 108) for the first 4 mo of age. A group of 88 breastfed infants served as a reference. During FU, anthropometrics were collected at 1, 3, 4, and 5 y of age, and blood pressure only at the last visit. RESULTS: Compared to Control, Concept group children had consistently lower mean BMI values during FU, with the most marked difference at 1 y of age (difference in means -0.71 kg/m2, 95% confidence interval (CI): -1.13, -0.29; P = 0.001); mean values were close to the breastfed group (P > 0.05). Contrary, the mean BMI values of the Control group were higher compared with the breastfed group during FU from 1 to 5 y of age (differences in means from 0.59 to 0.96 kg/m2, respectively; P < 0.02). At 5 y of age, the Concept group had a lower mean diastolic and arterial blood pressure compared with the Control group; -4.3mm Hg (95% CI: -7.3, -1.3; P = 0.005) and -3.7 mm Hg (95% CI: -6.5, -0.9; P = 0.01), respectively. CONCLUSIONS: Early life feeding of an innovative IMF with large, milk phospholipid-coated lipid droplets enriched with dairy lipids results in a BMI trajectory closer to breastfed infants and a lower blood pressure at school age. This trial was registered at the Dutch Trial Register as NTR3683 and NTR5538.

Identical IFT140 Variants Cause Variable Skeletal Ciliopathy Phenotypes-Challenges for the Accurate Diagnosis.

Ciliopathies are rare congenital disorders, caused by defects in the cilium, that cover a broad clinical spectrum. A subgroup of ciliopathies showing significant phenotypic overlap are known as skeletal ciliopathies and include Jeune asphyxiating thoracic dysplasia (JATD), Mainzer-Saldino syndrome (MZSDS), cranioectodermal dysplasia (CED), and short-rib polydactyly (SRP). Ciliopathies are heterogeneous disorders with >187 associated genes, of which some genes are described to cause more than one ciliopathy phenotype. Both the clinical and molecular overlap make accurate diagnosing of these disorders challenging. We describe two unrelated Polish patients presenting with a skeletal ciliopathy who share the same compound heterozygous variants in IFT140 (NM_014,714.4) r.2765_2768del; p.(Tyr923Leufs*28) and exon 27-30 duplication; p.(Tyr1152_Thr1394dup). Apart from overlapping clinical symptoms the patients also show phenotypic differences; patient 1 showed more resemblance to a Mainzer-Saldino syndrome (MZSDS) phenotype, while patient 2 was more similar to the phenotype of cranioectodermal dysplasia (CED). In addition, functional testing in patient-derived fibroblasts revealed a distinct cilium phenotyps for each patient, and strikingly, the cilium phenotype of CED-like patient 2 resembled that of known CED patients. Besides two variants in IFT140, in depth exome analysis of ciliopathy associated genes revealed a likely-pathogenic heterozygous variant in INTU for patient 2 that possibly affects the same IFT-A complex to which IFT140 belongs and thereby could add to the phenotype of patient 2. Taken together, by combining genetic data, functional test results, and clinical findings we were able to accurately diagnose patient 1 with "IFT140-related ciliopathy with MZSDS-like features" and patient 2 with "IFT140-related ciliopathy with CED-like features". This study emphasizes that identical variants in one ciliopathy associated gene can lead to a variable ciliopathy phenotype and that an in depth and integrated analysis of clinical, molecular and functional data is necessary to accurately diagnose ciliopathy patients.

The inhibitory Fc gamma IIb receptor dampens TLR4-mediated immune responses and is selectively up-regulated on dendritic cells from rheumatoid arthritis patients with quiescent disease.

Rheumatoid arthritis (RA) is a common autoimmune disease leading to profound disability and premature death. Although a role for FcgammaRs and TLRs is accepted, their precise involvement remains to be elucidated. FcgammaRIIb is an inhibitory FcR important in the maintenance of tolerance. We hypothesized that the inhibitory FcgammaRIIb inhibits TLR responses on monocyte-derived dendritic cells (DC) and serves as a counterregulatory mechanism to dampen inflammation, and we surmised that this mechanism might be defective in RA. The expression of the inhibitory FcgammaRIIb was found to be significantly higher on DCs from RA patients having low RA disease activity in the absence of treatment with antirheumatic drugs. The expression of activating FcgammaRs was similarly distributed among all RA patients and healthy controls. Intriguingly, only DCs with a high expression of FcgammaRIIb were able to inhibit TLR4-mediated secretion of proinflammatory cytokines when stimulated with immune complexes. In addition, when these DCs were coincubated with the combination of a TLR4 agonist and immune complexes, a markedly inhibited T cell proliferation was apparent, regulatory T cell development was promoted, and T cells were primed to produce high levels of IL-13 compared with stimulation of the DCs with the TLR4 agonist alone. Blocking FcgammaRIIb with specific Abs fully abrogated these effects demonstrating the full dependence on the inhibitory FcgammaRIIb in the induction of these phenomena. This TLR4-FcgammaRIIb interaction was shown to dependent on the PI3K and Akt pathway.

Cell-based assay for ciliopathy patients to improve accurate diagnosis using ALPACA.

Skeletal ciliopathies are a group of disorders caused by dysfunction of the cilium, a small signaling organelle present on nearly every vertebrate cell. This group of disorders is marked by genetic and clinical heterogeneity, which complicates accurate diagnosis. In this study, we developed a robust, standardized immunofluorescence approach to accurately diagnose a subset of these disorders. Hereto we determined and compared the cilium phenotype of healthy individuals to patients from three different ciliopathy subgroups, using skin-derived fibroblasts. The cilium phenotype assay consists of three parameters; (1) ciliogenesis, based on the presence or absence of cilium markers, (2) cilium length, measured by the combined signal of an axonemal and a cilium membrane marker, and (3) retrograde intraflagellar transport (IFT), quantified by the area of the ciliary tip. Analysis of the cilium phenotypic data yielded comparable and reproducible results and in addition, displayed identifiable clusters for healthy individuals and two ciliopathy subgroups, i.e. ATD and CED. Our results illustrate that standardized analysis of the cilium phenotype can be used to discriminate between ciliopathy subgroups. Therefore, we believe that standardization of functional assays analyzing cilium phenotypic data can provide additional proof for conclusive diagnosis of ciliopathies, which is essential for routine diagnostic care.