X-linked variations in SHROOM4 are implicated in congenital anomalies of the urinary tract and the anorectal, cardiovascular and central nervous systems.

BACKGROUND: SHROOM4 is thought to play an important role in cytoskeletal modification and development of the early nervous system. Previously, single-nucleotide variants (SNVs) or copy number variations (CNVs) in SHROOM4 have been associated with the neurodevelopmental disorder Stocco dos Santos syndrome, but not with congenital anomalies of the urinary tract and the visceral or the cardiovascular system. METHODS: Here, exome sequencing and CNV analyses besides expression studies in zebrafish and mouse and knockdown (KD) experiments using a splice blocking morpholino in zebrafish were performed to study the role of SHROOM4 during embryonic development. RESULTS: In this study, we identified putative disease-causing SNVs and CNVs in SHROOM4 in six individuals from four families with congenital anomalies of the urinary tract and the anorectal, cardiovascular and central nervous systems (CNS). Embryonic mouse and zebrafish expression studies showed Shroom4 expression in the upper and lower urinary tract, the developing cloaca, the heart and the cerebral CNS. KD studies in zebrafish larvae revealed pronephric cysts, anomalies of the cloaca and the heart, decreased eye-to-head ratio and higher mortality compared with controls. These phenotypes could be rescued by co-injection of human wild-type SHROOM4 mRNA and morpholino. CONCLUSION: The identified SNVs and CNVs in affected individuals with congenital anomalies of the urinary tract, the anorectal, the cardiovascular and the central nervous systems, and subsequent embryonic mouse and zebrafish studies suggest SHROOM4 as a developmental gene for different organ systems.

Regulation of Bone Morphogenetic Protein Receptor Type II Expression by FMR1/Fragile X Mental Retardation Protein in Human Granulosa Cells in the Context of Poor Ovarian Response.

Fragile X mental retardation protein (FMRP) is a translational repressor encoded by FMR1. It targets bone morphogenetic protein receptor type II (BMPR2), which regulates granulosa cell (GC) function and follicle development. However, whether this interaction affects folliculogenesis remains unclear. Therefore, this study investigated the potential effect of FMRP-BMPR2 dysregulation in ovarian reserves and infertility. COV434 cells and patient-derived GCs were used to evaluate FMRP and BMPR2 expression. Similarly, FMR1, BMPR2, LIMK1, and SMAD expression were evaluated in GCs with normal (NOR) and poor (POR) ovarian responses. FMRP and BMPR2 were expressed in both cell types. They were co-localized to the nuclear membrane of COV434 cells and cytoplasm of primary GCs. FMR1 silencing increased the mRNA and protein levels of BMPR2. However, the mRNA levels of FMR1 and BMPR2 were significantly lower in the POR group. FMR1 and BMPR2 levels were strongly positively correlated in the NOR group but weakly correlated in the POR group. Additionally, SMAD9 expression was significantly reduced in the POR group. This study highlights the crucial role of FMR1/FMRP in the regulation of BMPR2 expression and its impact on ovarian function. These findings indicate that the disruption of FMRP-BMPR2 interactions may cause poor ovarian responses and infertility.

[Genetic diagnostics and molecular approaches in pulmonary arterial hypertension]. / Genetische Diagnostik und molekulare Ansätze bei pulmonalarterieller Hypertonie.

The recently published new European guidelines for diagnosis and treatment of pulmonary hypertension now offer the so far most extensive description of genetic testing and counselling for pulmonary arterial hypertension patients. In addition, the importance of a clinical screening of healthy mutation carriers is highlighted as well as the genetic testing of patients with a suspicion of pulmonary veno-occlusive disease. We frame the respective parts of the guidelines on genetic testing and counselling in the context of recent data and provide comments. Finally, we give an outlook on novel molecular approaches starting from Sotatercept, addressing ion channels and novel therapeutic developments.

Gene panel diagnostics reveals new pathogenic variants in pulmonary arterial hypertension.

BACKGROUND: A genetic predisposition can lead to the rare disease pulmonary arterial hypertension (PAH). Most mutations have been identified in the gene BMPR2 in heritable PAH. However, as of today 15 further PAH genes have been described. The exact prevalence across these genes particularly in other PAH forms remains uncertain. We present the distribution of mutations across PAH genes identified at the largest German referral centre for genetic diagnostics in PAH over a course of > 3 years. METHODS: Our PAH-specific gene diagnostics panel was used to sequence 325 consecutive PAH patients from March 2017 to October 2020. For the first year the panel contained thirteen PAH genes: ACVRL1, BMPR1B, BMPR2, CAV1, EIF2AK4, ENG, GDF2, KCNA5, KCNK3, KLF2, SMAD4, SMAD9 and TBX4. These were extended by the three genes ATP13A3, AQP1 and SOX17 from March 2018 onwards following the genes' discovery. RESULTS: A total of 79 mutations were identified in 74 patients (23%). Of the variants 51 (65%) were located in the gene BMPR2 while the other 28 variants were found in ten further PAH genes. We identified disease-causing variants in the genes AQP1, KCNK3 and SOX17 in families with at least two PAH patients. Mutations were not only detected in patients with heritable and idiopathic but also with associated PAH. CONCLUSIONS: Genetic defects were identified in 23% of the patients in a total of 11 PAH genes. This illustrates the benefit of the specific gene panel containing all known PAH genes.

Activation of AKT/mammalian target of rapamycin signaling in the peripheral blood of women with premature ovarian insufficiency and its correlation with FMR1 expression.

BACKGROUND: The protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway regulates early follicular activation and follicular pool maintenance in female germline cells. Fragile X mental retardation 1 (FMR1) regulates folliculogenesis and it is variably expressed in patients with Premature Ovary Insufficiency. FMR1 expression is supposed to be linked to AKT/mTOR signaling in an ovarian response dependent manner as demonstrated in recent in vitro and in vivo studies in the female germline in vitro and in vivo. METHODS: We evaluated changes in the expression of AKT/mTOR signaling pathway genes by real time PCR in the peripheral blood of 74 patients with Premature Ovarian Insufficiency and 56 fertile controls and correlated their expression with FMR1 expression. RESULTS: Expression of the genes AKT1, TSC2, mTOR, and S6K was significantly more abundant in patients with POI than in the controls. For AKT1, TSC2 and mTOR, gene expression was not affected by FMR1-CGG repeat number in the 5´-untranslated region. FMR1 and S6K expression levels, however, were significantly upregulated in patients with POI and an FMR1 premutation. Independent of a premutation, expression of mTOR, S6K, and TSC2 was significantly correlated with that of FMR1 in all patients. Furthermore, when grouped according to ovarian reserve, this effect remained significant only for mTOR and S6K, with higher significance note in patients with Premature Ovarian Insufficiency than in the controls. CONCLUSIONS: In Premature ovarian insufficiency patients, activation of AKT/mTOR signaling pathway is remarkable and putatively pathognomonic. Additionally, it seems to be triggered by an FMR1/mTOR/S6K linkage mechanism, most relevant in premutation carriers.

Germ cell mosaicism for AUTS2 exon 6 deletion.

Haploinsufficiency of AUTS2 has been associated with neurodevelopmental disorders and dysmorphic features (MIM # 615834). More than 50 patients have been described, mostly carrying de novo deletions of one or more exons, including eight patients with exon 6 deletions. We report on two siblings, a girl and a boy aged 11 and 13 years, in whom the same pathogenic 85 kb deletion on 7q11.22 encompassing exon 6 of AUTS2 by SNP array analysis was identified. Both children had typical symptoms of AUTS2 syndrome such as intellectual impairment and behavioral problems, but with markedly different expression. SNP array analysis excluded the deletion in blood samples of both parents and a healthy brother. Conventional karyotyping of both parents and additional FISH analyses, marking the flanking regions of the deletion, did not show any structural rearrangements involving 7q11.22. A germ cell mosaicism was suggested as the most probable explanation for occurrence of the same deletion in these two siblings. To our knowledge this is the first report of germ cell mosaicism for AUTS2 syndrome. It additionally provides further evidence of intrafamilial phenotypic variability in AUTS2 syndrome and adds clinical information to the phenotypic spectrum of patients with AUTS2 exon 6 deletions.

A boy with Silver-Russell syndrome and Sotos syndrome.

Silver-Russell syndrome (SRS) is characterized by pre- and postnatal growth deficiency. It is most often caused by hypomethylation of the paternal imprinting center 1 of chromosome 11p15.5. In contrast, Sotos syndrome is an overgrowth syndrome that results either from pathogenic NSD1 gene variants or copy number variations affecting the NSD1 gene. Here, we report on a 6 month-old boy with severe short stature, relative macrocephaly, severe feeding difficulties with underweight, muscular hypotonia, motor delay, medullary nephrocalcinosis, bilateral sensorineural hearing impairment and facial dysmorphisms. SNP array revealed a 2.1 Mb de novo interstitial deletion of 5q35.2q35.3 encompassing the NSD1 gene. As Sotos syndrome could not satisfactorily explain his symptoms, diagnostic testing for SRS was initiated. It demonstrated hypomethylation of the imprinting center 1 of chromosome 11p15.5 confirming the clinically suspected SRS. We compared the symptoms of our patient with the typical clinical features of individuals with SRS and Sotos syndrome, respectively. To our knowledge, this is the first study reporting the very unusual coincidence of both Sotos syndrome and SRS in the same patient.

Quantitative retrospective natural history modeling for orphan drug development.

The natural history of most rare diseases is incompletely understood and usually relies on studies with low level of evidence. Consistent with the goals for future research of rare disease research set by the International Rare Diseases Research Consortium in 2017, the purpose of this paper is to review the recently developed method of quantitative retrospective natural history modeling (QUARNAM) and to illustrate its usefulness through didactically selected analyses examples in an overall population of 849 patients worldwide with seven (ultra-) rare neurogenetic disorders. A quantitative understanding of the natural history of the disease is fundamental for the development of specific interventions and counseling afflicted families. QUARNAM has a similar relationship to a published case study as a meta-analysis has to an individual published study. QUARNAM relies on sophisticated statistical analyses of published case reports focusing on four research questions: How long does it take to make the diagnosis? How long do patients live? Which factors predict disease severity (eg, genotypes, signs/symptoms, biomarkers)? Where can patients be recruited for studies? Useful statistical techniques include Kaplan-Meier estimates, cluster analysis, regression techniques, binary decisions trees, word clouds, and geographic mapping. In comparison to other natural history study methods (prospective studies or retrospective studies such as chart reviews), QUARNAM can provide fast information on hard clinical endpoints (ie, survival, diagnostic delay) with a lower effort. The choice of method for a particular drug development program may be driven by the research question and may encompass combinatory approaches.

Myeloproliferative Diseases as Possible Risk Factor for Development of Chronic Thromboembolic Pulmonary Hypertension-A Genetic Study.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare disease which is often caused by recurrent emboli. These are also frequently found in patients with myeloproliferative diseases. While myeloproliferative diseases can be caused by gene defects, the genetic predisposition to CTEPH is largely unexplored. Therefore, the objective of this study was to analyse these genes and further genes involved in pulmonary hypertension in CTEPH patients. A systematic screening was conducted for pathogenic variants using a gene panel based on next generation sequencing. CTEPH was diagnosed according to current guidelines. In this study, out of 40 CTEPH patients 4 (10%) carried pathogenic variants. One patient had a nonsense variant (c.2071A>T p.Lys691*) in the BMPR2 gene and three further patients carried the same pathogenic variant (missense variant, c.1849G>T p.Val617Phe) in the Janus kinase 2 (JAK2) gene. The latter led to a myeloproliferative disease in each patient. The prevalence of this JAK2 variant was significantly higher than expected (p < 0.0001). CTEPH patients may have a genetic predisposition more often than previously thought. The predisposition for myeloproliferative diseases could be an additional risk factor for CTEPH development. Thus, clinical screening for myeloproliferative diseases and genetic testing may be considered also for CTEPH patients.

Marker chromosomes can arise from chromothripsis and predict adverse prognosis in acute myeloid leukemia.

Metaphase karyotyping is an established diagnostic standard in acute myeloid leukemia (AML) for risk stratification. One of the cytogenetic findings in AML is structurally highly abnormal marker chromosomes. In this study, we have assessed frequency, cytogenetic characteristics, prognostic impact, and underlying biological origin of marker chromosomes. Given their inherent gross structural chromosomal damage, we speculated that they may arise from chromothripsis, a recently described phenomenon of chromosome fragmentation in a single catastrophic event. In 2 large consecutive prospective, randomized, multicenter, intensive chemotherapy trials (AML96, AML2003) from the Study Alliance Leukemia, marker chromosomes were detectable in 165/1026 (16.1%) of aberrant non-core-binding-factor (CBF) karyotype patients. Adverse-risk karyotypes displayed a higher frequency of marker chromosomes (26.5% in adverse-risk, 40.3% in complex aberrant, and 41.2% in abnormality(17p) karyotypes, P < .0001 each). Marker chromosomes were associated with a poorer prognosis compared with other non-CBF aberrant karyotypes and led to lower remission rates (complete remission + complete remission with incomplete recovery), inferior event-free survival as well as overall survival in both trials. In multivariate analysis, marker chromosomes independently predicted poor prognosis in the AML96 trial ≤60 years. As detected by array comparative genomic hybridization, about one-third of marker chromosomes (18/49) had arisen from chromothripsis, whereas this phenomenon was virtually undetectable in a control group of marker chromosome-negative complex aberrant karyotypes (1/34). The chromothripsis-positive cases were characterized by a particularly high degree of karyotype complexity, TP53 mutations, and dismal prognosis. In conclusion, marker chromosomes are indicative of chromothripsis and associated with poor prognosis per se and not merely by association with other adverse cytogenetic features.

FMR1 expression in human granulosa cells increases with exon 1 CGG repeat length depending on ovarian reserve.

BACKGROUND: Fragile-X-Mental-Retardation-1- (FMR1)-gene is supposed to be a key gene for ovarian reserve and folliculogenesis. It contains in its 5'-UTR a triplet-base-repeat (CGG), that varies between 26 and 34 in general population. CGG-repeat-lengths with 55-200 repeats (pre-mutation = PM) show instable heredity with a tendency to increase and are associated with premature-ovarian-insufficiency or failure (POI/POF) in about 20%. FMR1-mRNA-expression in leucocytes and granulosa cells (GCs) increases with CGG-repeat-length in PM-carriers, but variable FMR1-expression profiles were also described in women with POI without PM-FMR1 repeat-length. Additionally, associations between low numbers of retrieved oocytes and elevated FMR1-expression levels have been shown in GCs of females with mid-range PM-CGG-repeats without POI. Effects of FMR1-repeat-lengths-deviations (n < 26 or n > 34) below the PM range (n < 55) on ovarian reserve and response to ovarian stimulation remain controversial. METHODS: We enrolled 229 women undergoing controlled ovarian hyperstimulation for IVF/ICSI-treatment and devided them in three ovarian-response-subgroups: Poor responder (POR) after Bologna Criteria, polycystic ovary syndrome (PCO) after Rotterdam Criteria, or normal responder (NOR, control group). Subjects were subdivided into six genotypes according to their be-allelic CGG-repeat length. FMR1-CGG-repeat-length was determined using ALF-express-DNA-sequencer or ABI 3100/3130 × 1-sequencer. mRNA was extracted from GCs after follicular aspiration and quantitative FMR1-expression was determined using specific TaqMan-Assay and applying the ΔΔCT method. Kruskall-Wallis-Test or ANOVA were used for simple comparison between ovarian reserve (NOR, POR or PCO) and CGG-subgroups or cohort demographic data. All statistical analysis were performed with SPSS and statistical significance was set at p ≤ 0.05. RESULTS: A statistically significant increase in FMR1-mRNA-expression-levels was detected in GCs of PORs with heterozygous normal/low-CGG-repeat-length compared with other genotypes (p = 0.044). CONCLUSION: Female ovarian response may be negatively affected by low CGG-alleles during stimulation. In addition, due to a low-allele-effect, folliculogenesis may be impaired already prior to stimulation leading to diminished ovarian reserve and poor ovarian response. A better understanding of FMR1 expression-regulation in GCs may help to elucidate pathomechanisms of folliculogenesis disorders and to develop risk-adjusted treatments for IVF/ICSI-therapy. Herewith FMR1-genotyping potentially provides a better estimatation of treatment outcome and allows the optimal adaptation of stimulation protocols in future.

Impact of clinical exomes in neurodevelopmental and neurometabolic disorders.

Whole exome sequencing (WES) is well established in research and is now being introduced into clinically indicated diagnostics (so-called clinical exomes). We evaluated the diagnostic yield and clinical implications of WES in 72 patients from 60 families with undiagnosed neurodevelopmental disorders (NDD), neurometabolic disorders, and dystonias. Pathogenic or likely pathogenic variants leading to a molecular diagnosis could be identified in 21 of the 60 families (overall 35%, in 36% of patients with NDD, in 43% of patients with neurometabolic disorders, in 25% of patients with dystonias). In one family two coexisting autosomal recessive diseases caused by homozygous pathogenic variants in two different genes were diagnosed. In another family, a homozygous frameshift variant in STRADA was found to cause a severe NDD with early onset epilepsy, brain anomalies, hypotonia, heart defect, nephrocalcinosis, macrocephaly and distinctive facies so far designated as PMSE (polyhydramnios, megalencephaly, symptomatic epilepsy) syndrome. In 7 of the 21 families with a molecular diagnosis the pathogenic variants were only identified by clinical follow-up, manual reevaluation of the literature, a change of filter setting, and/or reconsideration of inheritance pattern. Most importantly, clinical implications included management changes in 8 cases and impact on family planning in 20 families with a molecular diagnosis. This study shows that reevaluation and follow-up can improve the diagnostic rate and that WES results have important implications on medical management and family planning. Furthermore, we could confirm STRADA as a gene associated with syndromic ID but find it questionable if the current designation as PMSE depicts the most important clinical features.

Novel recurrent chromosomal aberrations detected in clonal plasma cells of light chain amyloidosis patients show potential adverse prognostic effect: first results from a genome-wide copy number array analysis.

Immunoglobulin light chain (AL) amyloidosis is a rare plasma cell dyscrasia characterized by the deposition of abnormal amyloid fibrils in multiple organs, thus impairing their function. In the largest cohort studied up to now of 118 CD138-purified plasma cell samples from previously untreated immunoglobulin light chain amyloidosis patients, we assessed in parallel copy number alterations using high-density copy number arrays and interphase fluorescence in situ hybridization (iFISH). We used fluorescence in situ hybridization probes for the IgH translocations t(11;14), t(4;14), and t(14;16) or any other IgH rearrangement as well as numerical aberrations of the chromosome loci 1q21, 8p21, 5p15/5q35, 11q22.3 or 11q23, 13q14, 15q22, 17p13, and 19q13. Recurrent gains included chromosomes 1q (36%), 9 (24%), 11q (24%), as well as 19 (15%). Recurrent losses affected chromosome 13 (29% monosomy) and partial losses of 14q (19%), 16q (14%) and 13q (12%), respectively. In 88% of patients with translocation t(11;14), the hallmark chromosomal aberration in AL amyloidosis, a concomitant gain of 11q22.3/11q23 detected by iFISH was part of the unbalanced translocation der(14)t(11;14)(q13;q32) with the breakpoint in the CCND1/MYEOV gene region. Partial loss of chromosome regions 14q and 16q were significantly associated to gain 1q. Gain 1q21 detected by iFISH almost always resulted from a gain of the long arm of chromosome 1 and not from trisomy 1, whereas deletions on chromosome 1p were rarely found. Overall and event-free survival analysis found a potential adverse prognostic effect of concomitant gain 1q and deletion 14q as well as of deletion 1p. In conclusion, in the first whole genome report of clonal plasma cells in AL amyloidosis, novel aberrations and hitherto unknown potential adverse prognostic effects were uncovered.

First identification of Krüppel-like factor 2 mutation in heritable pulmonary arterial hypertension.

Heritable pulmonary arterial hypertension (HPAH) is an autosomal dominantly inherited disease caused by mutations in the bone morphogenic protein receptor 2 (BMPR2) gene and/or genes of its signalling pathway in approximately 85% of patients. We clinically and genetically analysed an HPAH family without mutations in previously described pulmonary arterial hypertension (PAH) genes. Clinical assessment included electrocardiogram, lung function, blood gas analysis, chest X-ray, laboratory testing, echocardiography and right heart catheterization in case of suspected disease. Genetic diagnostics were performed using a PAH-specific gene panel including all known 12 PAH genes and 20 further candidate genes by next-generation sequencing (NGS). HPAH was invasively confirmed in two sisters and their father who died aged 32 years. No signs of HPAH were detected in five first-degree family members. Both sisters were lung transplanted and remained stable during a follow-up of >20 years. We detected a novel missense mutation in the Krüppel-like factor 2 (KLF2) likely leading to a disruption of gene function. The same KLF2 mutation has been described as a recurrent somatic mutation in B-cell lymphoma. Neither the healthy family members carried the mutation nor >120000 controls. These findings point to KLF2 as a new PAH gene. Further studies are needed to assess frequency and implication of KLF2 mutations in PAH patients.

Diagnosis of CoPAN by whole exome sequencing: Waking up a sleeping tiger's eye.

Neurodegeneration with brain iron accumulation (NBIA) is a group of neurodegenerative disorders characterized by iron accumulation in the basal ganglia. Recently, mutations in CoA synthase (COASY) have been identified as a cause of a novel NBIA subtype (COASY Protein-Associated Neurodegeneration, CoPAN) in two patients with dystonic paraparesis, parkinsonian features, cognitive impairment, behavior abnormalities, and axonal neuropathy. COASY encodes an enzyme required for Coenzyme A (CoA) biosynthesis. Using whole exome sequencing (WES) we identified compound heterozygous COASY mutations in two siblings with intellectual disability, ataxic gait, progressive spasticity, and obsessive-compulsive behavior. The "eye-of-the tiger-sign," a characteristic hypointense spot within the hyperintense globi pallidi on MRI found in the most common subtype of NBIA (Pantothenate Kinase-Associated Neurodegeneration, PKAN), was not present. Instead, bilateral hyperintensity and swelling of caudate nucleus, putamen, and thalamus were found. In addition, our patients showed a small corpus callosum and frontotemporal and parietal white matter changes, expanding the brain phenotype of patients with CoPAN. Metabolic investigations showed increased free carnitine and decreased acylcarnitines in the patients dried blood samples. Carnitine palmitoyl transferase 1 (CPT1) deficiency was excluded by further enzymatic and metabolic investigations. As CoA and its derivate Acetyl-CoA play an essential role in fatty acid metabolism, we assume that abnormal acylcarnitine profiles are a result of the COASY mutations. This report not only illustrates that WES is a powerful tool to elucidate the etiology of rare genetic diseases, but also identifies unique neuroimaging and metabolic findings that may be key features for an early diagnosis of CoPAN.

DDX3X mutations in two girls with a phenotype overlapping Toriello-Carey syndrome.

Recently, de novo heterozygous variants in DDX3X have been reported in about 1.5% of 2659 females with previously unexplained intellectual disability (ID). We report on the identification of DDX3X variants in two unrelated girls with clinical features of Toriello-Carey Syndrome (T-CS). In patient 1, the recurrent variant c.1703C>T; p.(P568L) was identified when reconsidering X-linked de novo heterozygous variants in exome sequencing data. In patient 2, the DDX3X variant c.1600C>G; p.(R534G) was also detected by exome sequencing. Based on these data, de novo heterozygous DDX3X variants should be considered not only in females with unexplained ID, but also in individuals with a clinical diagnosis of T-CS.

FMR1 and AKT/mTOR signalling pathways: potential functional interactions controlling folliculogenesis in human granulosa cells.

Granulosa cells (GCs) play a major role in folliculogenesis and are crucial for oocyte maturation and growth. In these cells, the mTOR/AKT signalling pathway regulates early folliculogenesis by maintaining the dormancy of primordial follicles, while FSH induces their further differentiation and maturation. Because changes in number of CGG triplets in FMR1 exon 1 (below or beyond normal values of 26-34 triplets) affect ovarian reserve and pre-mutations containing >54 CGG triplets represent a known risk factor for premature ovarian insufficiency/failure, we investigated in the human GC model (COV434) how FMR1/FMRP and mTOR/AKT are expressed and potentially interact during GC proliferation. As FMR protein (FMRP) is expressed mainly in human ovarian GCs, we used these after inducing their proliferation using recombinant FSH (rFSH) and the repression of the mTOR/AKT signalling pathway. We showed that AKT and mTOR expression levels significantly increase after stimulation with rFSH, while S6K and FMR1 expression decrease. After inhibiting mTOR and AKT, FMR1 and S6K expression significantly increased. Only AKT inhibition led to decreased FMRP levels, as expected due to the known FMR1/FMRP negative feedback loop. But rFSH and the mTOR inhibition increased them, indicating a decoupling of this FMR1/FMRP negative feedback loop in our model system.

Critical appraisal of genotype assessment in molybdenum cofactor deficiency.

INTRODUCTION: Molybdenum cofactor deficiency (MoCD) is an ultra-orphan, life-threatening disease. Substrate substitution therapy has successfully been performed in single cases of MoCD type A and clinical trials are underway for drug registration. We present an innovative approach for classification of genotype severity to test the hypothesis that milder sequence variants in MoCD result in a less severe disease phenotype quantitated by patient survival. METHODS: All available worldwide published cases with clinical and genetic data were included (n = 40). We stratified the already published disease causing sequence variants as mild or severe with the use of in silico prediction programs, where possible and assessed the possible impact of the variants on the expression of the gene or function of the expressed protein. In a compound heterozygous situation the mildest sequence variant determined the genotype. Subsequently, clinical manifestations and outcomes of both groups were compared. RESULTS: Patients with a severe genotype showed a median survival of 15 months and had a lower probability of survival compared to patients with mild genotypes who were all alive at last reported follow-up (p = 0.0203, Log-rank test). DISCUSSION: The severity of the genotype assessed by in silico prediction and further classification explained survival in molybdenum cofactor deficiency and may therefore be considered a confounder for the outcome of therapeutic clinical trials requiring adjustment in the clinical trial design or analysis. These results should further be investigated by future in vitro or in vivo functional studies. Caution should be taken with this approach for the classification of variants in molecular genetic diagnostics or genetic counseling.

EIF2AK4 mutation as "second hit" in hereditary pulmonary arterial hypertension.

BACKGROUND: Mutations in the eukaryotic translation initiation factor 2α kinase 4 (EIF2AK4) gene have recently been identified in recessively inherited veno-occlusive disease. In this study we assessed if EIF2AK4 mutations occur also in a family with autosomal dominantly inherited pulmonary arterial hypertension (HPAH) and incomplete penetrance of bone morphogenic protein receptor 2 (BMPR2) mutations. METHODS: Clinical examinations in a family with 10 members included physical examination, electrocardiogram, (stress)-echocardiography and lung function. Manifest PAH was confirmed by right heart catheterisation in three affected subjects. Genetic analysis was performed using a new PAH-specific gene panel analysis with next generation sequencing of all known PAH and further candidate genes. Identified variants were confirmed by Sanger sequencing. RESULTS: All living family members with manifest HPAH carried two pathogenic heterozygous mutations: a frame shift mutation in the BMPR2 gene and a novel splice site mutation in the EIF2AK4 gene. Two family members who carried the BMPR2 mutation only did not develop manifest HPAH. CONCLUSIONS: This is the first study suggesting that EIF2AK4 can also contribute to autosomal dominantly inherited HPAH. Up to now it has only been identified in a recessive form of HPAH. Only those family members with a co-occurrence of two mutations developed manifest HPAH. Thus, the EIF2AK4 and BMRPR2 mutations support the "second hit" hypothesis explaining the variable penetrance of HPAH in this family. Hence, the assessment of all known PAH genes in families with a known mutation might assist in predictions about the clinical manifestation in so far non-affected mutation carriers.

Identification of genetic defects in pulmonary arterial hypertension by a new gene panel diagnostic tool.

In the present study we developed a new specific gene panel for pulmonary arterial hypertension (PAH) including major disease genes and further candidates. We assessed 37 patients with invasively confirmed PAH and five relatives of affected patients for genetic testing. A new PAH-specific gene panel was designed using next generation sequencing (NGS) including 12 known disease genes and 17 further candidates. Any potential pathogenic variants were reassessed by Sanger sequencing. Twenty-two of the 37 patients (59%) had a mutation in BMPR2, ALK1, ENG or EIF2AK4 genes identified by panel and Sanger sequencing. In addition, 12 unclassified variants were identified in seven genes (known and candidate genes). A sensitivity of 100% was met after quality parameters were adjusted. Specificity increased to 100% when Sanger technique was added as a routine validation. The new PAH-specific gene panel developed in the present study allowed for the first time the assessment of all known PAH genes and further candidates at once and markedly reduced overall sequencing time and costs. Sensitivity and specificity reached 100% when Sanger sequencing was additionally applied. Thus, this technique will potentially change the routine diagnostic genetic testing in PAH patients.