The Alazami Syndrome-Associated Protein LARP7 Guides U6 Small Nuclear RNA Modification and Contributes to Splicing Robustness.

The La-related protein 7 (LARP7) forms a complex with the nuclear 7SK RNA to regulate RNA polymerase II transcription. It has been implicated in cancer and the Alazami syndrome, a severe developmental disorder. Here, we report a so far unknown role of this protein in RNA modification. We show that LARP7 physically connects the spliceosomal U6 small nuclear RNA (snRNA) with a distinct subset of box C/D small nucleolar RNAs (snoRNAs) guiding U6 2'-O-methylation. Consistently, these modifications are severely compromised in the absence of LARP7. Although general splicing remains largely unaffected, transcriptome-wide analysis revealed perturbations in alternative splicing in LARP7-depleted cells. Importantly, we identified defects in 2'-O-methylation of the U6 snRNA in Alazami syndrome siblings carrying a LARP7 mutation. Our data identify LARP7 as a bridging factor for snoRNA-guided modification of the U6 snRNA and suggest that alterations in splicing fidelity contribute to the etiology of the Alazami syndrome.

A case report of Sanfilippo syndrome - the long way to diagnosis.

BACKGROUND: Mucopolysaccharidosis type III (Sanfilippo syndrome) is a lysosomal storage disorder, caused by a deficiency in the heparan-N-sulfatase enzyme involved in the catabolism of the glycosaminoglycan heparan sulfate. It is characterized by early nonspecific neuropsychiatric symptoms, followed by progressive neurocognitive impairment in combination with only mild somatic features. In this patient group with a broad clinical spectrum a significant genotype-phenotype correlation with some mutations leading to a slower progressive, attenuated course has been demonstrated. CASE PRESENTATION: Our patient had complications in the neonatal period and was diagnosed with Mucopolysaccharidosis IIIa only at the age of 28 years. He was compound heterozygous for the variants p.R245H and p.S298P, the latter having been shown to lead to a significantly milder phenotype. CONCLUSIONS: The diagnostic delay is even more prolonged in this patient population with comorbidities and a slowly progressive course of the disease.

Paternal 132 bp deletion affecting KCNQ1OT1 in 11p15.5 is associated with growth retardation but does not affect imprinting.

BACKGROUND: The chromosomal region 11p15.5 harbours two imprinting centres (H19/IGF2:IG-DMR/IC1, KCNQ1OT1:TSS-DMR/IC2). Molecular alterations of the IC2 are associated with Beckwith-Wiedemann syndrome (BWS), whereas only single patients with growth retardation and Silver-Russell syndrome (SRS) features have been reported. CNVs in 11p15.5 account for less than 1% of patients with BWS and SRS, and they mainly consist of duplications of both ICs either affecting the maternal (SRS) or the paternal (BWS) allele. However, this correlation does not apply to smaller CNVs, which are associated with diverse clinical outcomes. METHODS AND RESULTS: We identified a family with a 132 bp deletion within the KCNQ1OT1 gene, associated with growth retardation in case of paternal transmission but a normal phenotype when maternally inherited. Comparison of molecular and clinical data with cases from the literature helped to delineate its functional relevance. CONCLUSION: Microdeletions within the paternal IC2 affecting the KCNQ1OT1 gene have been described in only five families, and they all include the differentially methylated region KCNQ1OT1:TSS-DMR/IC2 and parts of the KCNQ1 gene. However, these deletions have different impacts on the expression of both genes and the cell-cycle inhibitor CDKN1C. They thereby cause different phenotypes. The 132 bp deletion is the smallest deletion in the IC2 reported so far. It does not affect the IC2 methylation in general and the coding sequence of the KCNQ1 gene. Thus, the deletion is only associated with a growth retardation phenotype when paternally transmitted but not with other clinical features in case of maternal inheritance as observed for larger deletions.

Heterogeneous phenotypes in families with duplications of the paternal allele within the imprinting center 1 (H19/IGF2:TSS-DMR) in 11p15.5.

The clinical impact of duplications affecting the 11p15.5 region is difficult to predict, and depends on the parent-of-origin of the affected allele as well as on the type (deletion, duplication), the extent and genomic content of the variant. Three unrelated families with inheritance of duplications affecting the IC1 region in 11p15.5 through two generations but different phenotypes (Beckwith-Wiedemann and Silver-Russell syndromes, normal phenotype) are reported. The inconsistent phenotypic patterns of carriers of the same variant strongly indicate the impact of cis- and/or trans-acting modifiers on the clinical outcome of IC1 duplication carriers.

Clinical relevance of systematic phenotyping and exome sequencing in patients with short stature.

PurposeShort stature is a common condition of great concern to patients and their families. Mostly genetic in origin, the underlying cause often remains elusive due to clinical and genetic heterogeneity.MethodsWe systematically phenotyped 565 patients where common nongenetic causes of short stature were excluded, selected 200 representative patients for whole-exome sequencing, and analyzed the identified variants for pathogenicity and the affected genes regarding their functional relevance for growth.ResultsBy standard targeted diagnostic and phenotype assessment, we identified a known disease cause in only 13.6% of the 565 patients. Whole-exome sequencing in 200 patients identified additional mutations in known short-stature genes in 16.5% of these patients who manifested only part of the symptomatology. In 15.5% of the 200 patients our findings were of significant clinical relevance. Heterozygous carriers of recessive skeletal dysplasia alleles represented 3.5% of the cases.ConclusionA combined approach of systematic phenotyping, targeted genetic testing, and whole-exome sequencing allows the identification of the underlying cause of short stature in at least 33% of cases, enabling physicians to improve diagnosis, treatment, and genetic counseling. Exome sequencing significantly increases the diagnostic yield and consequently care in patients with short stature.

Painful Charcot-Marie-Tooth neuropathy type 2E/1F due to a novel NEFL mutation.

INTRODUCTION: Charcot-Marie-Tooth neuropathy (CMT) 2E/1F is caused by mutations in the neurofilament light-chain polypeptide (NEFL) gene. Giant axons are a histological hallmark frequently seen in nerves of patients with CMT2E. METHODS: We describe the case of a 43-year-old patient with a painful, predominantly sensory neuropathy. RESULTS: The patient's sural nerve biopsy showed multiple giant axons. Genetic sequencing of the NEFL gene revealed that the patient was heterozygous for an altered sequence of the gene, c.816C>G, p.Asn272Lys, which has not yet been described in CMT2E/1F. CONCLUSION: In contrast to other cases of CMT2E/1F, where motor symptoms are predominant, pain was the most disabling symptom in this patient. Muscle Nerve 55: 752-755, 2017.

Rare copy number variants are a common cause of short stature.

Human growth has an estimated heritability of about 80%-90%. Nevertheless, the underlying cause of shortness of stature remains unknown in the majority of individuals. Genome-wide association studies (GWAS) showed that both common single nucleotide polymorphisms and copy number variants (CNVs) contribute to height variation under a polygenic model, although explaining only a small fraction of overall genetic variability in the general population. Under the hypothesis that severe forms of growth retardation might also be caused by major gene effects, we searched for rare CNVs in 200 families, 92 sporadic and 108 familial, with idiopathic short stature compared to 820 control individuals. Although similar in number, patients had overall significantly larger CNVs (p-value<1×10(-7)). In a gene-based analysis of all non-polymorphic CNVs>50 kb for gene function, tissue expression, and murine knock-out phenotypes, we identified 10 duplications and 10 deletions ranging in size from 109 kb to 14 Mb, of which 7 were de novo (p<0.03) and 13 inherited from the likewise affected parent but absent in controls. Patients with these likely disease causing 20 CNVs were smaller than the remaining group (p<0.01). Eleven (55%) of these CNVs either overlapped with known microaberration syndromes associated with short stature or contained GWAS loci for height. Haploinsufficiency (HI) score and further expression profiling suggested dosage sensitivity of major growth-related genes at these loci. Overall 10% of patients carried a disease-causing CNV indicating that, like in neurodevelopmental disorders, rare CNVs are a frequent cause of severe growth retardation.

Low-energy trauma-induced intercondylar femoral fracture.

We present a 44-year-old female patient with recurrent fragility fractures including an intercondylar femoral fracture and with normal planar bone densitometry. Diagnosis of hypophosphatasia was suggested by low volumetric cortical bone mineral density and laboratory findings. DNA sequencing revealed heterozygous mutations in the exons 5, 6 and 9 of the ALPL gene, thus confirming the suspected diagnosis.

Co-Occurence of Reciprocal Translocation and COL2A1 Mutation in a Fetus with Severe Skeletal Dysplasia: Implications for Genetic Counseling.

Achondrogenesis type II is an autosomal-dominant disease leading to severe micromelic dwarfism. Here, we report on the postmortem identification of a de novo heterozygous mutation in the COL2A1 gene (c.1529G>A, p.Gly510Asp) in a fetus who presented with generalized hydrops fetalis and severe micromelia during prenatal sonographic examinations. Initially, a reciprocal translocation t(4;17)(q31;p13) was detected in this fetus by chorionic villus sampling. Subsequent chromosomal analysis of maternal and paternal blood showed that the patient's mother was carrier of the same reciprocal translocation. SNP array analysis of the fetus did not provide evidence for chromosomal imbalances or CNVs that could be associated with the fetal phenotype. The coexistence of a cytogenetic (reciprocal translocation) and a molecular genetic (COL2A1 mutation) abnormality in the fetus carries important implications for genetic counseling.

Parkinson's disease in GTP cyclohydrolase 1 mutation carriers.

GTP cyclohydrolase 1, encoded by the GCH1 gene, is an essential enzyme for dopamine production in nigrostriatal cells. Loss-of-function mutations in GCH1 result in severe reduction of dopamine synthesis in nigrostriatal cells and are the most common cause of DOPA-responsive dystonia, a rare disease that classically presents in childhood with generalized dystonia and a dramatic long-lasting response to levodopa. We describe clinical, genetic and nigrostriatal dopaminergic imaging ([(123)I]N-ω-fluoropropyl-2ß-carbomethoxy-3ß-(4-iodophenyl) tropane single photon computed tomography) findings of four unrelated pedigrees with DOPA-responsive dystonia in which pathogenic GCH1 variants were identified in family members with adult-onset parkinsonism. Dopamine transporter imaging was abnormal in all parkinsonian patients, indicating Parkinson's disease-like nigrostriatal dopaminergic denervation. We subsequently explored the possibility that pathogenic GCH1 variants could contribute to the risk of developing Parkinson's disease, even in the absence of a family history for DOPA-responsive dystonia. The frequency of GCH1 variants was evaluated in whole-exome sequencing data of 1318 cases with Parkinson's disease and 5935 control subjects. Combining cases and controls, we identified a total of 11 different heterozygous GCH1 variants, all at low frequency. This list includes four pathogenic variants previously associated with DOPA-responsive dystonia (Q110X, V204I, K224R and M230I) and seven of undetermined clinical relevance (Q110E, T112A, A120S, D134G, I154V, R198Q and G217V). The frequency of GCH1 variants was significantly higher (Fisher's exact test P-value 0.0001) in cases (10/1318 = 0.75%) than in controls (6/5935 = 0.1%; odds ratio 7.5; 95% confidence interval 2.4-25.3). Our results show that rare GCH1 variants are associated with an increased risk for Parkinson's disease. These findings expand the clinical and biological relevance of GTP cycloydrolase 1 deficiency, suggesting that it not only leads to biochemical striatal dopamine depletion and DOPA-responsive dystonia, but also predisposes to nigrostriatal cell loss. Further insight into GCH1-associated pathogenetic mechanisms will shed light on the role of dopamine metabolism in nigral degeneration and Parkinson's disease.

New Insights on Genetic Diagnostics in Cardiomyopathy and Arrhythmia Patients Gained by Stepwise Exome Data Analysis.

Inherited cardiomyopathies are characterized by clinical and genetic heterogeneity that challenge genetic diagnostics. In this study, we examined the diagnostic benefit of exome data compared to targeted gene panel analyses, and we propose new candidate genes. We performed exome sequencing in a cohort of 61 consecutive patients with a diagnosis of cardiomyopathy or primary arrhythmia, and we analyzed the data following a stepwise approach. Overall, in 64% of patients, a variant of interest (VOI) was detected. The detection rate in the main sub-cohort consisting of patients with dilated cardiomyopathy (DCM) was much higher than previously reported (25/36; 69%). The majority of VOIs were found in disease-specific panels, while a further analysis of an extended panel and exome data led to an additional diagnostic yield of 13% and 5%, respectively. Exome data analysis also detected variants in candidate genes whose functional profile suggested a probable pathogenetic role, the strongest candidate being a truncating variant in STK38. In conclusion, although the diagnostic yield of gene panels is acceptable for routine diagnostics, the genetic heterogeneity of cardiomyopathies and the presence of still-unknown causes favor exome sequencing, which enables the detection of interesting phenotype-genotype correlations, as well as the identification of novel candidate genes.

Evolutionary conserved networks of human height identify multiple Mendelian causes of short stature.

Height is a heritable and highly heterogeneous trait. Short stature affects 3% of the population and in most cases is genetic in origin. After excluding known causes, 67% of affected individuals remain without diagnosis. To identify novel candidate genes for short stature, we performed exome sequencing in 254 unrelated families with short stature of unknown cause and identified variants in 63 candidate genes in 92 (36%) independent families. Based on systematic characterization of variants and functional analysis including expression in chondrocytes, we classified 13 genes as strong candidates. Whereas variants in at least two families were detected for all 13 candidates, two genes had variants in 6 (UBR4) and 8 (LAMA5) families, respectively. To facilitate their characterization, we established a clustered network of 1025 known growth and short stature genes, which yielded 29 significantly enriched clusters, including skeletal system development, appendage development, metabolic processes, and ciliopathy. Eleven of the candidate genes mapped to 21 of these clusters, including CPZ, EDEM3, FBRS, IFT81, KCND1, PLXNA3, RASA3, SLC7A8, UBR4, USP45, and ZFHX3. Fifty additional growth-related candidates we identified await confirmation in other affected families. Our study identifies Mendelian forms of growth retardation as an important component of idiopathic short stature.

Use of Targeted High-Throughput Sequencing for Genetic Classification of Patients with Bleeding Diathesis and Suspected Platelet Disorder.

Inherited platelet disorders (IPD) form a rare and heterogeneous disease entity that is present in about 8% of patients with non-acquired bleeding diathesis. Identification of the defective cellular pathway is an important criterion for stratifying the patient's individual risk profile and for choosing personalized therapeutic options. While costs of high-throughput sequencing technologies have rapidly declined over the last decade, molecular genetic diagnosis of bleeding and platelet disorders is getting more and more suitable within the diagnostic algorithms. In this study, we developed, verified, and evaluated a targeted, panel-based next-generation sequencing approach comprising 59 genes associated with IPD for a cohort of 38 patients with a history of recurrent bleeding episodes and functionally suspected, but so far genetically undefined IPD. DNA samples from five patients with genetically defined IPD with disease-causing variants in WAS , RBM8A , FERMT3 , P2YR12 , and MYH9 served as controls during the validation process. In 40% of 35 patients analyzed, we were able to finally detect 15 variants, eight of which were novel, in 11 genes, ACTN1 , AP3B1 , GFI1B , HPS1 , HPS4 , HPS6 , MPL , MYH9 , TBXA2R , TPM4 , and TUBB1 , and classified them according to current guidelines. Apart from seven variants of uncertain significance in 11% of patients, nine variants were classified as likely pathogenic or pathogenic providing a molecular diagnosis for 26% of patients. This report also emphasizes on potentials and pitfalls of this tool and prospectively proposes its rational implementation within the diagnostic algorithms of IPD.

Efficacy of anti-sclerostin monoclonal antibody BPS804 in adult patients with hypophosphatasia.

BACKGROUND: Hypophosphatasia (HPP) is a rare genetic disorder resulting in variable alterations of bone formation and mineralization that are caused by mutations in the ALPL gene, encoding the tissue-nonspecific alkaline phosphatase (ALP) enzyme. METHODS: In this phase IIA open-label, single-center, intra-patient, dose-escalating study, adult patients with HPP received 3 ascending intravenous doses of 5, 10, and 20 mg/kg BPS804, a fully human anti-sclerostin monoclonal antibody, on days 1, 15, and 29, respectively. Patients were followed for 16 weeks after the last dose. We assessed the pharmacodynamics, pharmacokinetics, preliminary efficacy, and safety of BPS804 administrations at specified intervals during treatment and follow-up. RESULTS: Eight patients (mean age 47.8 years) were enrolled in the study (6 females, 2 males). BPS804 treatment increased mean ALP and bone-specific ALP enzymatic activity between days 2 and 29. Transient increases in the bone formation markers procollagen type-I N-terminal propeptide (PINP), osteocalcin, and parathyroid hormone as well as a transient decrease in the bone resorption marker C-telopeptide of type I collagen (CTX-1) were observed. Lumbar spine bone mineral density showed a mean increase by day 85 and at end of study. Treatment-associated adverse events were mild and transient. CONCLUSION: BPS804 treatment was well tolerated and resulted in increases in bone formation biomarkers and bone mineral density, suggesting that sclerostin inhibition could be applied to enhance bone mineral density, stability, and regeneration in non-life-threatening clinical situations in adults with HPP. TRIAL REGISTRATION: Clinicaltrials.gov NCT01406977. FUNDING: Novartis Institutes for BioMedical Research, Basel, Switzerland.

ALS and MMN mimics in patients with BSCL2 mutations: the expanding clinical spectrum of SPG17 hereditary spastic paraplegia.

Silver syndrome/SPG17 is a motor manifestation of mutations in the BSCL2 gene and usually presents as a complicated form of hereditary spastic paraplegia (HSP). We present clinical data, follow-up, and genetic results of seven patients with Silver syndrome/SPG17 including a family with a variable intrafamilial phenotype ranging from subclinical signs to a severe and rapidly progressing amyotrophic lateral sclerosis (ALS)-like phenotype. For molecular diagnosis of the family, we used the TruSight Exome sequencing panel consisting of 2761 genes. We filtered for variants common to affected family members and for exclusive variants in the ALS-like index patient to find possible modifier mutations. We found that de novo mutations and/or incomplete penetrance in BSCL2 has been taken into account for Silver syndrome/SPG17 and confirm the large phenotypical heterogeneity of BSCL2 mutations. Our findings broaden the reported spectrum of the disease to an ALS-like and multifocal motor neuropathy phenotype and underline the need for further research for genetic modifiers due to the striking interindividual and intrafamilial variability.

Microsatellite instability of selective target genes in HNPCC-associated colon adenomas.

Microsatellite instability (MSI) occurs in most hereditary nonpolyposis colorectal cancers (HNPCC) and less frequently in sporadic tumors as the result of DNA mismatch repair (MMR) deficiency. Instability at coding microsatellites (cMS) in specific target genes causes frameshift mutations and functional inactivation of affected proteins, thereby providing a selective growth advantage to MMR deficient cells. At present, little is known about Selective Target Gene frameshift mutations in preneoplastic lesions. In this study, we examined 30 HNPCC-associated MSI-H colorectal adenomas of different grades of dysplasia for frameshift mutations in 26 cMS-bearing genes, which, according to our previous model, represent Selective Target genes of MSI. About 30% (8/26) of these genes showed a high mutation frequency (> or =50%) in colorectal adenomas, similar to the frequencies reported for colorectal carcinomas. Mutations in one gene (PTHL3) occurred significantly less frequently in MSI adenomas compared to published mutation rates in MSI carcinomas (36.0 vs 85.7%, P=0.023). Biallelic inactivation was observed in nine genes, thus emphasizing the functional impact of cMS instability on MSI tumorigenesis. Some genes showed a high frequency of frameshift mutations already at early stages of MSI colorectal tumorigenesis that increased with grade of dysplasia and transition to carcinoma. These include known Target Genes like BAX and TGFBR2, as well as three novel candidates, MACS, NDUFC2, and TAF1B. Overall, we have identified genes of potential relevance for the initiation and progression of MSI tumorigenesis, thus representing promising candidates for novel diagnostic and therapeutic approaches directed towards MMR-deficient tumors.

Absence of association between cyclin D1 (CCND1) G870A polymorphism and age of onset in hereditary nonpolyposis colorectal cancer.

CCND1 encodes cyclin D1, which plays an important role in the G1 to S phase transition of the cell cycle. A common polymorphism (c.G870A) increases alternate splicing. Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations in mismatch repair (MMR) genes, mainly MSH2 and MLH1, and shows a wide range in the age of its onset (AO), suggesting the existence of other modifying genetic factors. To date, two studies have investigated the association between CCND1 G/A variation and AO in HNPCC with contradictory results in 86 and 146 MMR mutation carriers, respectively. To clarify the role of the CCND1 G/A variation in HNPCC, we performed a study in 406 individuals carrying exclusively clear cut pathogenic mutations in MSH2 or MLH1. We did not observe a significant difference in genotype frequencies of affected and unaffected mutation carriers and healthy controls. A significant association between CCND1 genotypes and AO was found neither in the global comparison (log-rank, P = 0.2981; Wilcoxon, P = 0.2567) nor in a multivariate Cox regression analysis (hazard ratios 1.111, 95%CI 0.950-1.299, P = 0.188 and 1.090, 95%CI 0.868-1.369, P = 0.459 for the additive and dominant effect, respectively). We conclude, that the CCND1 G870A sequence variation is not a genetic modifier of the phenotype of HNPCC.