The role of genomic disorders in chronic kidney failure of undetermined aetiology ≤50 years.

Background: Genomic disorders caused by copy number variations (CNVs) are prevalent in patients with kidney disease; however, their contribution to chronic kidney failure (KF) of undetermined aetiology (uKF) is unclear. We screened patients with uKF aged 50 years or younger to establish the prevalence of causative CNVs. Methods: We enrolled patients with an onset of KF ≤50 years from suspected undetermined aetiology for initial review of medical records to exclude patients with clear-cut clinical or histopathological kidney diagnoses or patients with already established genetic kidney diseases. Next, we performed single nucleotide polymorphism (SNP) array-based CNV screening. All the detected CNVs were systematically classified and evaluated as possible causes of the patient's kidney disease. Patients with CNVs not explaining the kidney phenotype were additionally screened for causal variants in 540 genes using whole-genome sequencing. Results: We enrolled 172 patients, of whom 123 underwent SNP-array. Pathogenic CNVs corresponding to known genomic disorders were identified in 12 patients (9.8%). The identified genomic disorders provided a causative kidney diagnosis in three patients, all of whom had reached KF by age 18 years. The remaining nine patients had CNVs with unclear kidney disease causality. Subsequently, whole-genome sequencing provided a causative genetic diagnosis in an additional four patients, including two diagnostic sequence variants unrelated to the detected CNVs. Conclusions: Genomic disorders were prevalent in this cohort with uKF, and causative CNVs were identified in 5 of 123 patients. Further studies combining the analysis of CNVs and sequence variants are needed to clarify the causal role of genomic disorders in kidney disease.

[Incontinentia pigmenti in a newborn boy].

Incontinentia pigmenti is an uncommon X-linked dominant neurocutaneous ectodermal dysplasia. The disorder is usually lethal in males in utero, although it may occasionally occur in males with somatic mosaicsism or Klinefelter syndrome. This is a case report of a rare case of incontinentia pigmenti in a newborn male who presented with characteristic skin eruptions following Blaschko's lines. Histopathology and genetic testing confirmed the diagnosis. The management of patients with incontinentia pigmenti may require a multidisciplinary approach, and early diagnosis is of great importance.

Mosaicism for copy number variations in the placenta is even more difficult to interpret than mosaicism for whole chromosome aneuploidy.

OBJECTIVE: To compare mosaicisms in prenatal chorionic villus samples (CVSs) with corresponding postpartum placental samples. METHOD: We collected placentas from 15 consecutive cases of mosaicism detected in CVSs and obtained five standardized samples on each placenta after delivery. All pre- and postnatal placental samples were uncultured and analyzed by high-resolution chromosomal microarray. RESULTS: Ten cases of mosaicism for whole chromosome aneuploidy (mWC) and five cases with mosaicism for (sub)chromosomal copy number variations (mCNVs) were included. In 5/10 mWC cases and in 4/5 mCNV cases the prenatally detected aberration was confirmed in the postpartum placenta. Three postpartum placentas revealed various complex aberrations differing from the prenatal results: (1) mosaicisms for different deletions/duplications on 9p and 9q in all samples (prenatal: mosaic 5.3 Mb duplication on 9p24), (2) different regions with deletions/duplications/loss of heterozygosity on 1p in all samples (prenatal: mosaic 2.3 Mb 1p36 duplication), and (3) mosaicism for a duplication on 5q and a deletion on 6p in one out of five samples (prenatal: mosaic trisomy 7). CONCLUSION: CNVs constitute a complex subgroup in placental mosaicism. Counseling of these couples after chorionic villus sampling should not focus on the specific CNV involved, but on the nature of mosaicism and the option of amniocentesis and ultrasound.

14q32.11 microdeletion including CALM1, TTC7B, PSMC1, and RPS6KA5: A new potential cause of developmental and language delay in three unrelated patients.

Three unrelated patients with similar microdeletions of chromosome 14q32.11 with shared phenotypes including language and developmental delay, and four overlapping genes -CALM1, TTC7B, PSMC1, and RPS6KA5 have been presented. All four genes are expressed in the brain and have haploinsufficiency scores, which reflect low tolerance to loss of function variation. An insight on the genes in the overlapping region, which may influence the resulting phenotype has been provided. Given the three patients' similar phenotypes and lack of normal variation in this region, it was suggested that this microdeletion may be associated with developmental and language delay.

Perturbations in RhoA signalling cause altered migration and impaired neuritogenesis in human iPSC-derived neural cells with PARK2 mutation.

Mutations in parkin, encoded by the PARK2 gene, causes early-onset familial Parkinson's disease (PD), but dysfunctional parkin has also been implicated in sporadic PD. By combining human isogenic induced pluripotent stem cells (iPSCs) with and without PARK2 knockout (KO) and a novel large-scale mass spectrometry based proteomics and post-translational modification (PTM)-omics approach, we have mapped changes in protein profiles and PTMs caused by parkin deficiency in neurons. Our study identifies changes to several proteins previously shown to be dysregulated in brains of sporadic PD patients. Pathway analysis and subsequent in vitro assays reveal perturbations in migration and neurite outgrowth in the PARK2 KO neurons. We confirm the neurite defects using long-term engraftment of neurons in the striatum of immunosuppressed hemiparkinsonian adult rats. The GTP-binding protein RhoA was identified as a key upstream regulator, and RhoA activity was significantly increased in PARK2 KO neurons. By inhibiting RhoA signalling the migration and neurite outgrowth phenotypes could be rescued. Our study provides new insight into the pathogenesis of PD and demonstrates the broadly applicable potential of proteomics and PTMomics for elucidating the role of disease-causing mutations.

A case of penta X syndrome caused by nondisjunction in maternal meiosis 1 and 2.

The prenatal abnormalities in patients with penta X syndrome appear late in pregnancy and are nonspecific. In contrast, the postnatal phenotype is well described although new findings are still revealed. Penta X syndrome is a result of successive nondisjunctions of the X chromosomes in both maternal meiotic divisions.

Microarray-Based Analysis of Methylation of 1st Trimester Trisomic Placentas from Down Syndrome, Edwards Syndrome and Patau Syndrome.

Methylation-based non-invasive prenatal testing of fetal aneuploidies is an alternative method that could possibly improve fetal aneuploidy diagnosis, especially for trisomy 13(T13) and trisomy 18(T18). Our aim was to study the methylation landscape in placenta DNA from trisomy 13, 18 and 21 pregnancies in an attempt to find trisomy-specific methylation differences better suited for non-invasive prenatal diagnosis. We have conducted high-resolution methylation specific bead chip microarray analyses assessing more than 450,000 CpGs analyzing placentas from 12 T21 pregnancies, 12 T18 pregnancies and 6 T13 pregnancies. We have compared the methylation landscape of the trisomic placentas to the methylation landscape from normal placental DNA and to maternal blood cell DNA. Comparing trisomic placentas to normal placentas we identified 217 and 219 differentially methylated CpGs for CVS T18 and CVS T13, respectively (delta ß>0.2, FDR<0.05), but only three differentially methylated CpGs for T21. However, the methylation differences was only modest (delta ß<0.4), making them less suitable as diagnostic markers. Gene ontology enrichment analysis revealed that the gene set connected to theT18 differentially methylated CpGs was highly enriched for GO terms related to"DNA binding" and "transcription factor binding" coupled to the RNA polymerase II transcription. In the gene set connected to the T13 differentially methylated CpGs we found no significant enrichments.

17q12 deletion and duplication syndrome in Denmark-A clinical cohort of 38 patients and review of the literature.

17q12 deletions and duplications are two distinct, recurrent chromosomal aberrations usually diagnosed by chromosomal microarray analysis (CMA). The aberrations encompass the genes, HNF1B, LHX1, and ACACA, among others. We here describe a large national cohort of 12 phenotyped patients with 17q12 deletions and 26 phenotyped patients with 17q12 duplications. The total cohort includes 19 index patients and 19 family members. We also reviewed the literature in order to further improve the basis for the counseling. We emphasize that renal disease, learning disability, behavioral abnormalities, epilepsy, autism, schizophrenia, structural brain abnormalities, facial dysmorphism, and joint laxity are features seen in both the 17q12 deletion syndrome and the reciprocal 17q12 duplication syndrome; and we extend the list of features seen in both patient categories to include strabismus, esophageal defects, and duodenal atresia. Delayed language development, learning disability, kidney involvement, and eye dysmorphism and strabismus were the most consistently shared features among patients with 17q12 deletion. Patients with 17q12 duplications were characterized by an extremely wide phenotypic spectrum, including a variable degree of learning disabilities, delayed language development, delayed motor milestones, and a broad range of psychiatric and neurological features. This patient group also included adults achieving an academic degree. Assessing index patients and non-index patients separately, our observations illustrate that an overall milder disease burden is seen, in particular in patients with 17q12 duplications who are ascertained on the duplication rather than the phenotype. This evidence may be useful in prenatal counseling. © 2016 Wiley Periodicals, Inc.

Microarray-Based Analysis of Methylation Status of CpGs in Placental DNA and Maternal Blood DNA--Potential New Epigenetic Biomarkers for Cell Free Fetal DNA-Based Diagnosis.

Epigenetic markers for cell free fetal DNA in the maternal blood circulation are highly interesting in the field of non-invasive prenatal testing since such markers will offer a possibility to quantify the amount of fetal DNA derived from different chromosomes in a maternal blood sample. The aim of the present study was to define new fetal specific epigenetic markers present in placental DNA that can be utilized in non-invasive prenatal diagnosis. We have conducted a high-resolution methylation specific beadchip microarray study assessing more than 450.000 CpG sites. We have analyzed the DNA methylation profiles of 10 maternal blood samples and compared them to 12 1st trimesters chorionic samples from normal placentas, identifying a number of CpG sites that are differentially methylated in maternal blood cells compared to chorionic tissue. To strengthen the utility of these differentially methylated CpG sites to be used with methyl-sensitive restriction enzymes (MSRE) in PCR-based NIPD, we furthermore refined the list of selected sites, containing a restriction sites for one of 16 different methylation-sensitive restriction enzymes. We present a list of markers on chromosomes 13, 18 and 21 with a potential for aneuploidy testing as well as a list of markers for regions harboring sub-microscopic deletion- or duplication syndromes.

Dosage changes of a segment at 17p13.1 lead to intellectual disability and microcephaly as a result of complex genetic interaction of multiple genes.

The 17p13.1 microdeletion syndrome is a recently described genomic disorder with a core clinical phenotype of intellectual disability, poor to absent speech, dysmorphic features, and a constellation of more variable clinical features, most prominently microcephaly. We identified five subjects with copy-number variants (CNVs) on 17p13.1 for whom we performed detailed clinical and molecular studies. Breakpoint mapping and retrospective analysis of published cases refined the smallest region of overlap (SRO) for microcephaly to a genomic interval containing nine genes. Dissection of this phenotype in zebrafish embryos revealed a complex genetic architecture: dosage perturbation of four genes (ASGR1, ACADVL, DVL2, and GABARAP) impeded neurodevelopment and decreased dosage of the same loci caused a reduced mitotic index in vitro. Moreover, epistatic analyses in vivo showed that dosage perturbations of discrete gene pairings induce microcephaly. Taken together, these studies support a model in which concomitant dosage perturbation of multiple genes within the CNV drive the microcephaly and possibly other neurodevelopmental phenotypes associated with rearrangements in the 17p13.1 SRO.

Duplication in the microtubule-actin cross-linking factor 1 gene causes a novel neuromuscular condition.

Spectrins and plakins are important communicators linking cytoskeletal components to each other and to cellular junctions. Microtubule-actin cross-linking factor 1 (MACF1) belongs to the spectraplakin family and is involved in control of microtubule dynamics. Complete knock out of MACF1 in mice is associated with developmental retardation and embryonic lethality. Here we present a family with a novel neuromuscular condition. Genetic analyses show a heterozygous duplication resulting in reduced MACF1 gene product. The functional consequence is affected motility observed as periodic hypotonia, lax muscles and diminished motor skills, with heterogeneous presentation among the affected family members. To corroborate these findings we used RNA interference to knock down the VAB-10 locus containing the MACF1 homologue in C. elegans, and we could show that this also causes movement disturbances. These findings suggest that changes in the MACF1 gene is implicated in this neuromuscular condition, which is an important observation since MACF1 has not previously been associated with any human disease and thus presents a key to understanding the essential nature of this gene.

Defining the phenotype associated with microduplication reciprocal to Sotos syndrome microdeletion.

NSD1 point mutations, submicroscopic deletions and intragenic deletions are the major cause of Sotos syndrome, characterized by pre-postnatal generalized overgrowth with advanced bone age, learning disability, seizures, distinctive facial phenotype. Reverse clinical phenotype due to 5q35 microduplication encompassing NSD1 gene has been reported so far in 27 cases presenting with delayed bone age, microcephaly, failure to thrive and seizures in some cases, further supporting a gene dosage effect of NSD1 on growth regulation and neurological functions. Here we depict the clinical presentation of three new cases with 5q35 microduplication outlining a novel syndrome characterized by microcephaly, short stature, developmental delay and in some cases delayed bone maturation, without any typical facial or osseous anomalies.

The duplication 17p13.3 phenotype: analysis of 21 families delineates developmental, behavioral and brain abnormalities, and rare variant phenotypes.

Chromosome 17p13.3 is a gene rich region that when deleted is associated with the well-known Miller-Dieker syndrome. A recently described duplication syndrome involving this region has been associated with intellectual impairment, autism and occasional brain MRI abnormalities. We report 34 additional patients from 21 families to further delineate the clinical, neurological, behavioral, and brain imaging findings. We found a highly diverse phenotype with inter- and intrafamilial variability, especially in cognitive development. The most specific phenotype occurred in individuals with large duplications that include both the YWHAE and LIS1 genes. These patients had a relatively distinct facial phenotype and frequent structural brain abnormalities involving the corpus callosum, cerebellar vermis, and cranial base. Autism spectrum disorders were seen in a third of duplication probands, most commonly in those with duplications of YWHAE and flanking genes such as CRK. The typical neurobehavioral phenotype was usually seen in those with the larger duplications. We did not confirm the association of early overgrowth with involvement of YWHAE and CRK, or growth failure with duplications of LIS1. Older patients were often overweight. Three variant phenotypes included cleft lip/palate (CLP), split hand/foot with long bone deficiency (SHFLD), and a connective tissue phenotype resembling Marfan syndrome. The duplications in patients with clefts appear to disrupt ABR, while the SHFLD phenotype was associated with duplication of BHLHA9 as noted in two recent reports. The connective tissue phenotype did not have a convincing critical region. Our experience with this large cohort expands knowledge of this diverse duplication syndrome.

Age-dependence of relative telomere length profiles during spermatogenesis in man.

Telomeres, the protective structures at the outmost ends of chromosomes, shorten in all somatic cells with each cell-division and by cumulative oxidative damage. To counteract that these shortened telomeres are passed on to offspring, the telomeres are elongated by the enzyme, telomerase, during human spermatogenesis. A few groups have tried to elucidate this process by measuring telomerase activity in the various cell-types during spermatogenesis, but until now, no one has ever measured telomere length (TL) during these different stages in humans. Some groups have measured TL in spermatozoa and surprisingly found that telomeres of older men are longer, than those from younger men. To elucidate this phenomenon we investigated if the distribution of TL over the various precursor germ cells in old males differed from young males, perhaps indicating a more ubiquitous telomere elongation in testes from older men. We therefore obtained testicular biopsies from 6 older and 6 younger men undergoing vasectomy. The cells were suspended as single cells and smeared onto slides, followed by characterization of cell stages by phase contrast microscopy. Mean TL in individual cells was subsequently measured by telomere QFISH. Our data revealed no difference in the TL profile during spermatogenesis between younger and older men. All men had a similar profile which strongly resembled the telomerase expression profile found by others. This indicates that the longer telomeres in older men are not caused by a wider window of telomere elongation, stretching over more cell-types of spermatogenesis.

Heart defects and other features of the 22q11 distal deletion syndrome.

22q11.2 distal deletion syndrome is distinct from the common 22q11.2 deletion syndrome and caused by microdeletions localized adjacent to the common 22q11 deletion at its telomeric end. Most distal deletions of 22q11 extend from LCR22-4 to an LCR in the range LCR22-5 to LCR22-8. We present three patients with 22q11 distal deletions, of whom two have complex congenital heart malformation, thus broadening the phenotypic spectrum. We compare cardiac malformations reported in 22q11 distal deletion to those reported in the common 22q11 deletion syndrome. We also review the literature for patients with 22q11 distal deletions, and discuss the possible roles of haploinsufficiency of the MAPK1 gene. We find the most frequent features in 22q11 distal deletion to be developmental delay or learning disability, short stature, microcephalus, premature birth with low birth weight, and congenital heart malformation ranging from minor anomalies to complex malformations. Behavioral problems are also seen in a substantial portion of patients. The following dysmorphic features are relatively common: smooth philtrum, abnormally structured ears, cleft palate/bifid uvula, micro-/retrognathia, upslanting palpebral fissures, thin upper lip, and ear tags. Very distal deletions including region LCR22-6 to LCR22-7 encompassing the SMARCB1-gene are associated with an increased risk of malignant rhabdoid tumors.

The distribution pattern of critically short telomeres in human osteoarthritic knees.

INTRODUCTION: Telomere shortening is associated with a number of common age-related diseases. A role of telomere shortening in osteoarthritis (OA) has been suggested, mainly based on the assessment of mean telomere length in ex vivo expanded chondrocytes. We addressed this role directly in vivo by using a newly developed assay, which measures specifically the load of ultra-short single telomeres (below 1,500 base pairs), that is, the telomere subpopulation believed to promote cellular senescence. METHODS: Samples were obtained from human OA knees at two distances from the central lesion site. Each sample was split into three: one was used for quantification of ultra-short single telomeres through the Universal single telomere length assay (STELA), one for histological Mankin grading of OA, and one for mean telomere length measurement through quantitative fluorescence in situ hybridization (Q-FISH) as well as for assessment of senescence through quantification of senescence-associated heterochromatin foci (SAHF). RESULTS: The load of ultra-short telomeres as well as mean telomere length was significantly associated with proximity to lesions, OA severity, and senescence level. The degree of significance was higher when assessed through load of ultra-short telomeres per cell compared with mean telomere length. CONCLUSIONS: These in vivo data, especially the quantification of ultra-short telomeres, stress a role of telomere shortening in human OA.

Further delineation of 17p13.3 microdeletion involving CRK. The effect of growth hormone treatment.

Recently, a 17p13.3 microdeletion syndrome characterized by significant postnatal growth retardation, mild to moderate mental retardation and facial dysmorphic manifestations has been delineated to a small region within the area of the Miller-Dieker syndrome critical region. We report a boy with a 284 kb deletion within the Miller-Dieker critical region including CRK, but not involving YWHAE and TUSC5. He showed mental retardation and had significant postnatal growth retardation. Further, he had slight facial and limb abnormalities. Cerebral MRI, including visualization of the pituitary gland, disclosed no abnormalities. The findings in the present case indicate, that CRK may also be involved in the facial phenotype of the 17p13.3 microdeletion syndrome, and that CRK, and not YWHAE, seems to be involved in limb malformations. The effect of growth hormone treatment in CRK-deficient children is discussed.