Germline C1GALT1C1 mutation causes a multisystem chaperonopathy.

Mutations in genes encoding molecular chaperones can lead to chaperonopathies, but none have so far been identified causing congenital disorders of glycosylation. Here we identified two maternal half-brothers with a novel chaperonopathy, causing impaired protein O-glycosylation. The patients have a decreased activity of T-synthase (C1GALT1), an enzyme that exclusively synthesizes the T-antigen, a ubiquitous O-glycan core structure and precursor for all extended O-glycans. The T-synthase function is dependent on its specific molecular chaperone Cosmc, which is encoded by X-chromosomal C1GALT1C1. Both patients carry the hemizygous variant c.59C>A (p.Ala20Asp; A20D-Cosmc) in C1GALT1C1. They exhibit developmental delay, immunodeficiency, short stature, thrombocytopenia, and acute kidney injury (AKI) resembling atypical hemolytic uremic syndrome. Their heterozygous mother and maternal grandmother show an attenuated phenotype with skewed X-inactivation in blood. AKI in the male patients proved fully responsive to treatment with the complement inhibitor Eculizumab. This germline variant occurs within the transmembrane domain of Cosmc, resulting in dramatically reduced expression of the Cosmc protein. Although A20D-Cosmc is functional, its decreased expression, though in a cell or tissue-specific manner, causes a large reduction of T-synthase protein and activity, which accordingly leads to expression of varied amounts of pathological Tn-antigen (GalNAcα1-O-Ser/Thr/Tyr) on multiple glycoproteins. Transient transfection of patient lymphoblastoid cells with wild-type C1GALT1C1 partially rescued the T-synthase and glycosylation defect. Interestingly, all four affected individuals have high levels of galactose-deficient IgA1 in sera. These results demonstrate that the A20D-Cosmc mutation defines a novel O-glycan chaperonopathy and causes the altered O-glycosylation status in these patients.

O'Donnell-Luria-Rodan syndrome: description of a second multinational cohort and refinement of the phenotypic spectrum.

BACKGROUND: O'Donnell-Luria-Rodan syndrome (ODLURO) is an autosomal-dominant neurodevelopmental disorder caused by pathogenic, mostly truncating variants in KMT2E. It was first described by O'Donnell-Luria et al in 2019 in a cohort of 38 patients. Clinical features encompass macrocephaly, mild intellectual disability (ID), autism spectrum disorder (ASD) susceptibility and seizure susceptibility. METHODS: Affected individuals were ascertained at paediatric and genetic centres in various countries by diagnostic chromosome microarray or exome/genome sequencing. Patients were collected into a case cohort and were systematically phenotyped where possible. RESULTS: We report 18 additional patients from 17 families with genetically confirmed ODLURO. We identified 15 different heterozygous likely pathogenic or pathogenic sequence variants (14 novel) and two partial microdeletions of KMT2E. We confirm and refine the phenotypic spectrum of the KMT2E-related neurodevelopmental disorder, especially concerning cognitive development, with rather mild ID and macrocephaly with subtle facial features in most patients. We observe a high prevalence of ASD in our cohort (41%), while seizures are present in only two patients. We extend the phenotypic spectrum by sleep disturbances. CONCLUSION: Our study, bringing the total of known patients with ODLURO to more than 60 within 2 years of the first publication, suggests an unexpectedly high relative frequency of this syndrome worldwide. It seems likely that ODLURO, although just recently described, is among the more common single-gene aetiologies of neurodevelopmental delay and ASD. We present the second systematic case series of patients with ODLURO, further refining the mutational and phenotypic spectrum of this not-so-rare syndrome.

The Discovery of a LEMD2-Associated Nuclear Envelopathy with Early Progeroid Appearance Suggests Advanced Applications for AI-Driven Facial Phenotyping.

Over a relatively short period of time, the clinical geneticist's "toolbox" has been expanded by machine-learning algorithms for image analysis, which can be applied to the task of syndrome identification on the basis of facial photographs, but these technologies harbor potential beyond the recognition of established phenotypes. Here, we comprehensively characterized two individuals with a hitherto unknown genetic disorder caused by the same de novo mutation in LEMD2 (c.1436C>T;p.Ser479Phe), the gene which encodes the nuclear envelope protein LEM domain-containing protein 2 (LEMD2). Despite different ages and ethnic backgrounds, both individuals share a progeria-like facial phenotype and a distinct combination of physical and neurologic anomalies, such as growth retardation; hypoplastic jaws crowded with multiple supernumerary, yet unerupted, teeth; and cerebellar intention tremor. Immunofluorescence analyses of patient fibroblasts revealed mutation-induced disturbance of nuclear architecture, recapitulating previously published data in LEMD2-deficient cell lines, and additional experiments suggested mislocalization of mutant LEMD2 protein within the nuclear lamina. Computational analysis of facial features with two different deep neural networks showed phenotypic proximity to other nuclear envelopathies. One of the algorithms, when trained to recognize syndromic similarity (rather than specific syndromes) in an unsupervised approach, clustered both individuals closely together, providing hypothesis-free hints for a common genetic etiology. We show that a recurrent de novo mutation in LEMD2 causes a nuclear envelopathy whose prognosis in adolescence is relatively good in comparison to that of classical Hutchinson-Gilford progeria syndrome, and we suggest that the application of artificial intelligence to the analysis of patient images can facilitate the discovery of new genetic disorders.

Mutations in PIGB Cause an Inherited GPI Biosynthesis Defect with an Axonal Neuropathy and Metabolic Abnormality in Severe Cases.

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.

Alport syndrome and autosomal dominant tubulointerstitial kidney disease frequently underlie end-stage renal disease of unknown origin-a single-center analysis.

BACKGROUND: The prevalence of end-stage renal disease of unknown etiology in adult patients is globally high and accounts for almost 20% of all dialysis patients. Recent studies have suggested that the percentage of adult patients with a causal genetic variant has been underestimated so far. Despite severe prognostic and therapeutic implications, awareness about prevalence and manifestations of genetic kidney diseases in adult renal patients is still limited. METHODS: We recruited 58 individuals from 39 families at our transplantation center, fulfilling at least one of the following criteria: (i) unclear etiology of kidney disease, (ii) clinically suspected genetic kidney disease and (iii) positive family history for nephropathies. The cohort consisted of patients waitlisted for kidney transplantation and patients in the follow-up after transplantation. Detailed documentation of family history and phenotype was obtained before initiating gene panel sequencing of 479 nephropathy-associated genes. RESULTS: With this study design, a molecular genetic diagnosis was established in one-third of all patients. Mutations in the collagen COL4A genes, and mutations in MUC1 and UMOD were the most frequent among all detected causal variants. Overall, rare genetic variants were detected in more than half of all cases. CONCLUSION: The combination of detailed phenotyping prior to next-generation sequencing diagnostics was highly efficient. Elucidating the underlying genetic causes in a cohort of adult renal patients has considerable clinical impact on medical management.

Refining genotype-phenotype correlations in 304 patients with autosomal recessive polycystic kidney disease and PKHD1 gene variants.

Autosomal recessive polycystic kidney disease (ARPKD) is a severe disease of early childhood that is clinically characterized by fibrocystic changes of the kidneys and the liver. The main cause of ARPKD are variants in the PKHD1 gene encoding the large transmembrane protein fibrocystin. The mechanisms underlying the observed clinical heterogeneity in ARPKD remain incompletely understood, partly due to the fact that genotype-phenotype correlations have been limited to the association of biallelic null variants in PKHD1 with the most severe phenotypes. In this observational study we analyzed a deep clinical dataset of 304 patients with ARPKD from two independent cohorts and identified novel genotype-phenotype correlations during childhood and adolescence. Biallelic null variants frequently show severe courses. Additionally, our data suggest that the affected region in PKHD1 is important in determining the phenotype. Patients with two missense variants affecting amino acids 709-1837 of fibrocystin or a missense variant in this region and a null variant less frequently developed chronic kidney failure, and patients with missense variants affecting amino acids 1838-2624 showed better hepatic outcome. Variants affecting amino acids 2625-4074 of fibrocystin were associated with poorer hepatic outcome. Thus, our data expand the understanding of genotype-phenotype correlations in pediatric ARPKD patients and can lay the foundation for more precise and personalized counselling and treatment approaches.

Variants in PRKAR1B cause a neurodevelopmental disorder with autism spectrum disorder, apraxia, and insensitivity to pain.

PURPOSE: We characterize the clinical and molecular phenotypes of six unrelated individuals with intellectual disability and autism spectrum disorder who carry heterozygous missense variants of the PRKAR1B gene, which encodes the R1ß subunit of the cyclic AMP-dependent protein kinase A (PKA). METHODS: Variants of PRKAR1B were identified by single- or trio-exome analysis. We contacted the families and physicians of the six individuals to collect phenotypic information, performed in vitro analyses of the identified PRKAR1B-variants, and investigated PRKAR1B expression during embryonic development. RESULTS: Recent studies of large patient cohorts with neurodevelopmental disorders found significant enrichment of de novo missense variants in PRKAR1B. In our cohort, de novo origin of the PRKAR1B variants could be confirmed in five of six individuals, and four carried the same heterozygous de novo variant c.1003C>T (p.Arg335Trp; NM_001164760). Global developmental delay, autism spectrum disorder, and apraxia/dyspraxia have been reported in all six, and reduced pain sensitivity was found in three individuals carrying the c.1003C>T variant. PRKAR1B expression in the brain was demonstrated during human embryonal development. Additionally, in vitro analyses revealed altered basal PKA activity in cells transfected with variant-harboring PRKAR1B expression constructs. CONCLUSION: Our study provides strong evidence for a PRKAR1B-related neurodevelopmental disorder.

Long-term data on two sisters with C3GN due to an identical, homozygous CFH mutation and autoantibodies.

C3 glomerulonephritis (C3GN) is a rare but severe form of kidney disease caused by fluid-phase dysregulation of the alternative complement pathway. Causative mutations in complement regulating genes as well as auto-immune forms of C3GN have been described. However, therapy and prognosis in individual patients remain a matter of debate and long-term data are scarce. This also applies for the management of transplant patients as disease recurrence post-transplant is frequent. Here, we depict the clinical courses of two sisters with the unique combination of an identical, homozygous mutation in the complement factor H (CFH) gene as well as autoantibodies with a clinical follow-up of more than 20 years. Interestingly, the sisters presented with discordant clinical courses of C3GN with normal kidney function in one (patient A) and end-stage kidney disease in the other sister (patient B). In patient B, eculizumab was administered immediately prior to and in the course after kidney transplantation, with the result of a stable graft function without any signs of disease recurrence. Comprehensive genetic work-up revealed no further disease-causing mutation in both sisters. Intriguingly, the auto-antibody profile substantially differed in both sisters: autoantibodies in patient A reduced the C3b deposition, while the antibodies identified in patient B increased complement activation and deposition of split products. This study underlines the concept of a personalized-medicine approach in complement-associated diseases after thorough evaluation of the individual risk profile in each patient.

Patients with primary hyperoxaluria type 2 have significant morbidity and require careful follow-up.

Primary hyperoxaluria type 2 is a rare inherited disorder of glyoxylate metabolism causing nephrocalcinosis, renal stone formation and ultimately kidney failure. Previously, primary hyperoxaluria type 2 was considered to have a more favorable prognosis than primary hyperoxaluria type 1, but earlier reports are limited by low patient numbers and short follow up periods. Here we report on the clinical, genetic, and biochemical findings from the largest cohort of patients with primary hyperoxaluria type 2, obtained by a retrospective record review of genetically confirmed cases in the OxalEurope registry, a dataset containing 101 patients from eleven countries. Median follow up was 12.4 years. Median ages at first symptom and diagnosis for index cases were 3.2 years and 8.0 years, respectively. Urolithiasis was the most common presenting feature (82.8% of patients). Genetic analysis revealed 18 novel mutations in the GRHPR gene. Of 238 spot-urine analyses, 23 (9.7%) were within the normal range for oxalate as compared to less than 4% of 24-hour urine collections. Median intra-individual variation of 24-hour oxalate excretion was substantial (34.1%). At time of review, 12 patients were lost to follow-up; 45 of the remaining 89 patients experienced chronic kidney disease stage 2 or greater and 22 patients had reached stage 5. Median renal survival was 43.3 years, including 15 kidney transplantations in 11 patients (1 combined with liver transplantation). Renal outcome did not correlate with genotype, biochemical parameters or initially present nephrocalcinosis. Thus, primary hyperoxaluria type 2 is a disease with significant morbidity. Accurate diagnosis by 24-hour urine analysis and genetic testing are required with careful follow-up.

Which genes to assess in the NGS diagnostics of intellectual disability? The case for a consensus database-driven and expert-curated approach.

When deciding on which genes to assess in larger Next-Generation Sequencing (NGS) datasets for the molecular genetic diagnosis of intellectual disability (ID), geneticists today have a variety of gene-phenotype databases and expert-curated gene lists available. To quantify their respective completeness, we compare an ID gene selection auto-generated from the Human Phenotype Ontology gene-phenotype association database and expert-curated ID gene lists from three reputable sources (sysID, the DDD consortium and Genomics England) and analyse some of their differences. We give examples of what we regard as genuine gaps ("missing ID genes") for each of these and conclude that a complementary or consensus approach is needed to maximise diagnostic yield in ID patients. We propose several consensus gene lists with ID-associated genes of different confidence levels.

Risk Factors for Early Dialysis Dependency in Autosomal Recessive Polycystic Kidney Disease.

OBJECTIVE: To identify prenatal, perinatal, and postnatal risk factors for dialysis within the first year of life in children with autosomal recessive polycystic kidney disease (ARPKD) as a basis for parental counseling after prenatal and perinatal diagnosis. STUDY DESIGN: A dataset comprising 385 patients from the ARegPKD international registry study was analyzed for potential risk markers for dialysis during the first year of life. RESULTS: Thirty-six out of 385 children (9.4%) commenced dialysis in the first year of life. According to multivariable Cox regression analysis, the presence of oligohydramnios or anhydramnios, prenatal kidney enlargement, a low Apgar score, and the need for postnatal breathing support were independently associated with an increased hazard ratio for requiring dialysis within the first year of life. The increased risk associated with Apgar score and perinatal assisted breathing was time-dependent and vanished after 5 and 8 months of life, respectively. The predicted probabilities for early dialysis varied from 1.5% (95% CI, 0.5%-4.1%) for patients with ARPKD with no prenatal sonographic abnormalities to 32.3% (95% CI, 22.2%-44.5%) in cases of documented oligohydramnios or anhydramnios, renal cysts, and enlarged kidneys. CONCLUSIONS: This study, which identified risk factors associated with onset of dialysis in ARPKD in the first year of life, may be helpful in prenatal parental counseling in cases of suspected ARPKD.

A heritable microduplication encompassing TBL1XR1 causes a genomic sister-disorder for the 3q26.32 microdeletion syndrome.

Recently, a new syndrome with intellectual disability (ID) and dysmorphic features due to deletions or point mutations within the TBL1XR1 gene located in the chromosomal band 3q26.32 has been described (MRD41, OMIM 616944). One recurrent point mutation in the TBL1XR1 gene has been identified as the cause of Pierpont syndrome (OMIM 602342), a distinct intellectual disability syndrome with plantar lipomatosis. In addition, different de novo point mutations in the TBL1XR1 gene have been found in patients with autism spectrum disorders (ASD) and intellectual disability. Here, we report four patients from two unrelated families in whom array-CGH analysis and real-time quantitative PCR of genomic DNA revealed a TBL1XR1-microduplication. Adjacent genes were not affected. The microduplication occurred as a de novo event in one patient, whereas the other three cases occurred in two generations of a second, unrelated family. We compare and contrast the clinical findings in TBL1XR1 microdeletion, point mutation, and microduplication cases and expand the TBL1XR1-associated phenotypic spectrum. ID, hearing loss, and ASD are common features of TBL1XR1-associated diseases. Our clinical observations add to the increasing evidence of the role of TBL1XR1 in brain development, and they simultaneously demonstrate that different genetic disease mechanisms affecting TBL1XR1 can lead to similar ID phenotypes. The TBL1XR1-microduplication syndrome is an intellectual disability/learning disability syndrome with associated incomplete penetrance ASD, hearing loss, and delay of puberty. Its phenotypic overlap indicates that it is a genomic sister-disorder to the 3q26.32 microdeletion syndrome.

Prenatal ultrasound, genotype, and outcome in a large cohort of prenatally affected patients with autosomal-recessive polycystic kidney disease and other hereditary cystic kidney diseases.

PURPOSE: To investigate the sonographic and clinical genotype-phenotype correlations in autosomal recessive polycystic kidney disease (ARPKD) and other cystic kidney diseases (CKD) in a large cohort of prenatally detected fetuses with hereditary CKD. METHODS: We retrospectively studied the clinical and diagnostic data of 398 patients referred with prenatal ultrasound findings suggestive of CKD between 1994 and 2010. Cases with confirmed hereditary CKD (n = 130) were analyzed as to their prenatal ultrasound findings, genotype, and possible predictors of clinical outcome. RESULTS: ARPKD was most common in our non-representative sample. Truncating PKHD1 mutations led to a significantly reduced neonatal prognosis, with two such mutations being invariably lethal. Sonographically visible kidney cysts occurred in only 3% of ARPKD cases. Renal abnormalities in Meckel syndrome (MKS) appeared earlier than in ADPKD (19.6 ± 3.7 vs. 29.8 ± 5.1 GW) or ARPKD (19.6 ± 3.7 vs. 30.2 ± 1.2 GW). Additional CNS malformations were not found in ARPKD, but were highly sensitive for MKS. Pulmonary hypoplasia, oligo/anhydramnios (OAH), and kidney enlargement were associated with a significantly worse neonatal prognosis. CONCLUSION: Genotype, sonographic signs of OAH, enlarged kidney size, and pulmonary hypoplasia can be useful predictors of neonatal survival. We propose sonographic morphological criteria for ARPKD, ADPKD, MKS, and renal cyst and diabetes syndrome (RCAD). We further propose a clinical diagnostic algorithm for differentiating cystic kidney diseases.

Features of Postpartum Hemorrhage-Associated Thrombotic Microangiopathy and Role of Short-Term Complement Inhibition.

Introduction: In pregnancy-related atypical hemolytic uremic syndrome (p-aHUS), transferring recommendations for treatment decisions from nonpregnant cohorts with thrombotic microangiopathy (TMA) is difficult. Although potential causes of p-aHUS may be unrelated to inherent complement defects, peripartal complications such as postpartum hemorrhage (PPH) or (pre)eclampsia or Hemolysis, Elevated Liver enzymes and Low Platelets (HELLP) syndrome may be unrecognized drivers of complement activation. Methods: To evaluate diagnostic and therapeutic decisions in the practical real-life setting, we conducted an analysis of a cohort of 40 patients from 3 German academic hospitals with a diagnosis of p-aHUS, stratified by the presence (n = 25) or absence (n = 15) of PPH. Results: Histological signs of TMA were observed in 84.2% of all patients (100% vs. 72.7% in patients without or with PPH, respectively). Patients without PPH had a higher likelihood (20% vs. 0%) of pathogenic genetic abnormalities in the complement system although notably less than in other published cohorts. Four of 5 patients with observed renal cortical necrosis (RCN) after PPH received complement inhibition and experienced partially recovered kidney function. Patients on complement inhibition with or without PPH had an increased need for kidney replacement therapy (KRT) and plasma exchange (PEX). Because renal recovery was comparable among all patients treated with complement inhibition, a potential beneficial effect in this group of pregnancy-associated TMAs and p-aHUS is presumed. Conclusion: Based on our findings, we suggest a pragmatic approach toward limited and short-term anticomplement therapy for patients with a clinical diagnosis of p-aHUS, which should be stopped once causes of TMA other than genetic complement abnormalities emerge.

A Low-Cost Sequencing Platform for Rapid Genotyping in ADPKD and its Impact on Clinical Care.

Introduction: Autosomal-dominant polycystic kidney disease (ADPKD) is the most common genetic cause of kidney failure. Because of the heterogeneity in disease progression in ADPKD, parameters predicting future outcome are important. The disease-causing genetic variant is one of these parameters. Methods: A multiplex polymerase chain reaction (PCR)-based panel (MPP) was established for analysis of 6 polycystic kidney disease (PKD) genes (PKD1, PKD2, HNF1B, GANAB, DZIP1L, and PKHD1) in 441 patients with ADPKD. Selected patients were additionally sequenced using Sanger sequencing or a custom enrichment-based gene panel. Results were combined with clinical characteristics to assess the impact of genetic data on clinical decision-making. Variants of unclear significance (VUS) were considered diagnostic based on a classic ADPKD clinical phenotype. Results: Using the MPP, disease-causing variants were detected in 65.3% of patients. Sanger sequencing and the custom gene panel in 32 patients who were MPP-negative revealed 20 variants missed by MPP, (estimated overall false negative rate 24.6%, false-positive rate 9.4%). Combining clinical and genetic data revealed that knowledge of the genotype could have impacted the treatment decision in 8.2% of patients with a molecular genetic diagnosis. Sequencing only the PKD1 pseudogene homologous region in MPP-negative patients resulted in an acceptable false-negative rate of 3.28%. Conclusion: The MPP yields rapid genotype information at lower costs and allows for simple extension of the panel for new disease genes. Additional sequencing of the PKD1 pseudogene homologous region is required in negative cases. Access to genotype information even in settings with limited resources is important to allow for optimal patient counseling in ADPKD.

Copeptin in autosomal dominant polycystic kidney disease: real-world experiences from a large prospective cohort study.

Background: The identification of new biomarkers in autosomal-dominant polycystic kidney disease (ADPKD) is crucial to improve and simplify prognostic assessment as a basis for patient selection for targeted therapies. Post hoc analyses of the TEMPO 3:4 study indicated that copeptin could be one of those biomarkers. Methods: Copeptin was tested in serum samples from patients of the AD(H)PKD study. Serum copeptin levels were measured using a time-resolved amplified cryptate emission (TRACE)-based assay. In total, we collected 711 values from 389 patients without tolvaptan treatment and a total of 243 values (of which 64 were pre-tolvaptan) from 94 patients on tolvaptan. These were associated with rapid progression and disease-causing gene variants and their predictive capacity tested and compared with the Mayo Classification. Results: As expected, copeptin levels showed a significant negative correlation with estimated glomerular filtration rate (eGFR). Measurements on tolvaptan showed significantly higher copeptin levels (9.871 pmol/L vs 23.90 pmol/L at 90/30 mg; P < .0001) in all chronic kidney disease stages. Linear regression models (n = 133) show that copeptin is an independent predictor of eGFR slope. A clinical model (including eGFR, age, gender, copeptin) was nearly as good (R2 = 0.1196) as our optimal model (including height-adjusted total kidney volume, eGFR, copeptin, R2 = 0.1256). Adding copeptin to the Mayo model improved future eGFR estimation. Conclusion: Copeptin levels are associated with kidney function and independently explained future eGFR slopes. As expected, treatment with tolvaptan strongly increases copeptin levels.

Long-read sequencing identifies a common transposition haplotype predisposing for CLCNKB deletions.

BACKGROUND: Long-read sequencing is increasingly used to uncover structural variants in the human genome, both functionally neutral and deleterious. Structural variants occur more frequently in regions with a high homology or repetitive segments, and one rearrangement may predispose to additional events. Bartter syndrome type 3 (BS 3) is a monogenic tubulopathy caused by deleterious variants in the chloride channel gene CLCNKB, a high proportion of these being large gene deletions. Multiplex ligation-dependent probe amplification, the current diagnostic gold standard for this type of mutation, will indicate a simple homozygous gene deletion in biallelic deletion carriers. However, since the phenotypic spectrum of BS 3 is broad even among biallelic deletion carriers, we undertook a more detailed analysis of precise breakpoint regions and genomic structure. METHODS: Structural variants in 32 BS 3 patients from 29 families and one BS4b patient with CLCNKB deletions were investigated using long-read and synthetic long-read sequencing, as well as targeted long-read sequencing approaches. RESULTS: We report a ~3 kb duplication of 3'-UTR CLCNKB material transposed to the corresponding locus of the neighbouring CLCNKA gene, also found on ~50 % of alleles in healthy control individuals. This previously unknown common haplotype is significantly enriched in our cohort of patients with CLCNKB deletions (45 of 51 alleles with haplotype information, 2.2 kb and 3.0 kb transposition taken together, p=9.16×10-9). Breakpoint coordinates for the CLCNKB deletion were identifiable in 28 patients, with three being compound heterozygous. In total, eight different alleles were found, one of them a complex rearrangement with three breakpoint regions. Two patients had different CLCNKA/CLCNKB hybrid genes encoding a predicted CLCNKA/CLCNKB hybrid protein with likely residual function. CONCLUSIONS: The presence of multiple different deletion alleles in our cohort suggests that large CLCNKB gene deletions originated from many independently recurring genomic events clustered in a few hot spots. The uncovered associated sequence transposition haplotype apparently predisposes to these additional events. The spectrum of CLCNKB deletion alleles is broader than expected and likely still incomplete, but represents an obvious candidate for future genotype/phenotype association studies. We suggest a sensitive and cost-efficient approach, consisting of indirect sequence capture and long-read sequencing, to analyse disease-relevant structural variant hotspots in general.

Unraveling Structural Rearrangements of the CFH Gene Cluster in Atypical Hemolytic Uremic Syndrome Patients Using Molecular Combing and Long-Fragment Targeted Sequencing.

Complement factor H (CFH) and its related proteins have an essential role in regulating the alternative pathway of the complement system. Mutations and structural variants (SVs) of the CFH gene cluster, consisting of CFH and its five related genes (CFHR1-5), have been reported in renal pathologies as well as in complex immune diseases like age-related macular degeneration and systemic lupus erythematosus. SV analysis of this cluster is challenging because of its high degree of sequence homology. Following first-line next-generation sequencing gene panel sequencing, we applied Genomic Vision's Molecular Combing Technology to detect and visualize SVs within the CFH gene cluster and resolve its structural haplotypes completely. This approach was tested in three patients with atypical hemolytic uremic syndrome and known SVs and 18 patients with atypical hemolytic uremic syndrome or complement factor 3 glomerulopathy with unknown CFH gene cluster haplotypes. Three SVs, a CFH/CFHR1 hybrid gene in two patients and a rare heterozygous CFHR4/CFHR1 deletion in trans with the common CFHR3/CFHR1 deletion in a third patient, were newly identified. For the latter, the breakpoints were determined using a targeted enrichment approach for long DNA fragments (Samplix Xdrop) in combination with Oxford Nanopore sequencing. Molecular combing in addition to next-generation sequencing was able to improve the molecular genetic yield in this pilot study. This (cost-)effective approach warrants validation in larger cohorts with CFH/CFHR-associated disease.

Carrier testing for autosomal recessive disorders: a look at current practice in Germany.

Counseling recurrence risks for monogenic disorders is one of the mainstays of human genetics. However, in practice, consultations concerning autosomal recessive disorders exceed the simple conveyance of a 25 % recurrence risk for future offspring. Medical geneticists should be aware of the multifaceted way in which autosomal recessive disorders can pose a diagnostic and counseling challenge in their daily lives and of the pitfalls they might encounter. Although the intentional or incidental detection of carrier states for autosomal recessive diseases happens more and more frequently, our current practice when clarifying their associated reproductive risks remains unsystematic and often subjectively guided. We question whether the approach of focusing on small recurrence risks for a single familial disease with extensive single-gene tests in the partner of a known carrier truly addresses the counseling needs of a couple seeking preconceptional genetic advice. Different perspectives between patients and medical practitioners (or between different medical practitioners) on "acceptable risks" or the extent to which such risks must be minimized raise the question of whether existing professional guidelines need to be clarified.

Expanding the Spectrum of FAT1 Nephropathies by Novel Mutations That Affect Hippo Signaling.

INTRODUCTION: Disease-causing mutations in the protocadherin FAT1 have been recently described both in patients with a glomerulotubular nephropathy and in patients with a syndromic nephropathy. METHODS: We identified 4 patients with FAT1-associated disease, performed clinical and genetic characterization, and compared our findings to the previously published patients. Patient-derived primary urinary epithelial cells were analyzed by quantitative polymerase chain reaction (qPCR) and immunoblotting to identify possible alterations in Hippo signaling. RESULTS: Here we expand the spectrum of FAT1-associated disease with the identification of novel FAT1 mutations in 4 patients from 3 families (homozygous truncating variants in 3, compound heterozygous missense variants in 1 patient). All patients show an ophthalmologic phenotype together with heterogeneous renal phenotypes ranging from normal renal function to early-onset end-stage kidney failure. Molecular analysis of primary urine-derived urinary renal epithelial cells revealed alterations in the Hippo signaling cascade with a decreased phosphorylation of both the core kinase MST and the downstream effector YAP. Consistently, we found a transcriptional upregulation of bona fide YAP target genes. CONCLUSION: A comprehensive review of the here identified patients and those previously published indicates a highly diverse phenotype in patients with missense mutations but a more uniform and better recognizable phenotype in the patients with truncating mutations. Altered Hippo signaling and de-repressed YAP activity might be novel contributing factors to the pathomechanism in FAT1-associated renal disease.