The Phenotypic Continuum of ATP1A3-Related Disorders.

BACKGROUND AND OBJECTIVES: ATP1A3 is associated with a broad spectrum of predominantly neurologic disorders, which continues to expand beyond the initially defined phenotypes of alternating hemiplegia of childhood, rapid-onset dystonia parkinsonism, and cerebellar ataxia, areflexia, pes cavus, optic atrophy, sensorineural hearing loss syndrome. This phenotypic variability makes it challenging to assess the pathogenicity of an ATP1A3 variant found in an undiagnosed patient. We describe the phenotypic features of individuals carrying a pathogenic/likely pathogenic ATP1A3 variant and perform a literature review of all ATP1A3 variants published thus far in association with human neurologic disease. Our aim is to demonstrate the heterogeneous clinical spectrum of the gene and look for phenotypic overlap between patients that will streamline the diagnostic process. METHODS: Undiagnosed individuals with ATP1A3 variants were identified within the cohort of the Deciphering Developmental Disorders study with additional cases contributed by collaborators internationally. Detailed clinical data were collected with consent through a questionnaire completed by the referring clinicians. PubMed was searched for publications containing the term "ATP1A3" from 2004 to 2021. RESULTS: Twenty-four individuals with a previously undiagnosed neurologic phenotype were found to carry 21 ATP1A3 variants. Eight variants have been previously published. Patients experienced on average 2-3 different types of paroxysmal events. Permanent neurologic features were common including microcephaly (7; 29%), ataxia (13; 54%), dystonia (10; 42%), and hypotonia (7; 29%). All patients had cognitive impairment. Neuropsychiatric diagnoses were reported in 16 (66.6%) individuals. Phenotypes were extremely varied, and most individuals did not fit clinical criteria for previously published phenotypes. On review of the literature, 1,108 individuals have been reported carrying 168 different ATP1A3 variants. The most common variants are associated with well-defined phenotypes, while more rare variants often result in very rare symptom correlations, such as are seen in our study. Combined Annotation-Dependent Depletion (CADD) scores of pathogenic and likely pathogenic variants were significantly higher and variants clustered within 6 regions of constraint. DISCUSSION: Our study shows that looking for a combination of paroxysmal events, hyperkinesia, neuropsychiatric symptoms, and cognitive impairment and evaluating the CADD score and variant location can help identify an ATP1A3-related condition, rather than applying diagnostic criteria alone.

Sacral abnormalities including caudal appendage, skeletal dysplasia, and prenatal cardiomyopathy associated with a pathogenic TAB2 variant in a 3-generation family.

Haplo-insufficiency of the TGFß-activated kinase 1 binding protein 2 (TAB2) gene is associated with short stature, facial dysmorphisms, connective tissue abnormalities, hearing loss, and cardiac disease. Skeletal dysplasia and sacral dimples are also found in a minority of patients. Here, we describe a 3-generation family with caudal appendage, other sacral anomalies, and skeletal abnormalities including hypoplasia of the iliac wings and scapulae, fusion of the carpal bones and stenosis of the spinal canal, as well as a remarkable course of prenatally-detected cardiomyopathy with characteristics changing over time. Genetic analysis showed a heterozygous nonsense variant in the TAB2 gene.

Diagnostic Value of a Protocolized In-Depth Evaluation of Pediatric Bone Marrow Failure: A Multi-Center Prospective Cohort Study.

Background: Severe multilineage cytopenia in childhood caused by bone marrow failure (BMF) often represents a serious condition requiring specific management. Patients are at risk for invasive infections and bleeding complications. Previous studies report low rates of identifiable causes of pediatric BMF, rendering most patients with a descriptive diagnosis such as aplastic anemia (AA). Methods: We conducted a multi-center prospective cohort study in which an extensive diagnostic approach for pediatric patients with suspected BMF was implemented. After exclusion of malignant and transient causes of BMF, patients entered thorough diagnostic evaluation including bone marrow analysis, whole exome sequencing (WES) including copy number variation (CNV) analysis and/or single nucleotide polymorphisms (SNP) array analysis. In addition, functional and immunological evaluation were performed. Here we report the outcomes of the first 50 patients (2017-2021) evaluated by this approach. Results: In 20 patients (40%) a causative diagnosis was made. In this group, 18 diagnoses were established by genetic analysis, including 14 mutations and 4 chromosomal deletions. The 2 remaining patients had short telomeres while no causative genetic defect was found. Of the remaining 30 patients (60%), 21 were diagnosed with severe aplastic anemia (SAA) based on peripheral multi-lineage cytopenia and hypoplastic bone marrow, and 9 were classified as unexplained cytopenia without bone marrow hypoplasia. In total 28 patients had undergone hematopoietic stem cell transplantation (HSCT) of which 22 patients with an unknown cause and 6 patients with an identified cause for BMF. Conclusion: We conclude that a standardized in-depth diagnostic protocol as presented here, can increase the frequency of identifiable causes within the heterogeneous group of pediatric BMF. We underline the importance of full genetic analysis complemented by functional tests of all patients as genetic causes are not limited to patients with typical (syndromal) clinical characteristics beyond cytopenia. In addition, it is of importance to apply genome wide genetic analysis, since defects in novel genes are frequently discovered in this group. Identification of a causal abnormality consequently has implications for the choice of treatment and in some cases prevention of invasive therapies.

Biallelic Variants in the Ectonucleotidase ENTPD1 Cause a Complex Neurodevelopmental Disorder with Intellectual Disability, Distinct White Matter Abnormalities, and Spastic Paraplegia.

OBJECTIVE: Human genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with frequently only few affected individuals described for each gene. Herein, we characterize a large cohort of individuals with biallelic variation in ENTPD1, a gene previously linked to spastic paraplegia 64 (Mendelian Inheritance in Man # 615683). METHODS: Individuals with biallelic ENTPD1 variants were recruited worldwide. Deep phenotyping and molecular characterization were performed. RESULTS: A total of 27 individuals from 17 unrelated families were studied; additional phenotypic information was collected from published cases. Twelve novel pathogenic ENTPD1 variants are described (NM 001776.6): c.398_399delinsAA; p.(Gly133Glu), c.540del; p.(Thr181Leufs*18), c.640del; p.(Gly216Glufs*75), c.185 T > G; p.(Leu62*), c.1531 T > C; p.(*511Glnext*100), c.967C > T; p.(Gln323*), c.414-2_414-1del, and c.146 A > G; p.(Tyr49Cys) including 4 recurrent variants c.1109 T > A; p.(Leu370*), c.574-6_574-3del, c.770_771del; p.(Gly257Glufs*18), and c.1041del; p.(Ile348Phefs*19). Shared disease traits include childhood onset, progressive spastic paraplegia, intellectual disability (ID), dysarthria, and white matter abnormalities. In vitro assays demonstrate that ENTPD1 expression and function are impaired and that c.574-6_574-3del causes exon skipping. Global metabolomics demonstrate ENTPD1 deficiency leads to impaired nucleotide, lipid, and energy metabolism. INTERPRETATION: The ENTPD1 locus trait consists of childhood disease onset, ID, progressive spastic paraparesis, dysarthria, dysmorphisms, and white matter abnormalities, with some individuals showing neurocognitive regression. Investigation of an allelic series of ENTPD1 (1) expands previously described features of ENTPD1-related neurological disease, (2) highlights the importance of genotype-driven deep phenotyping, (3) documents the need for global collaborative efforts to characterize rare autosomal recessive disease traits, and (4) provides insights into disease trait neurobiology. ANN NEUROL 2022;92:304-321.

Biallelic ADAM22 pathogenic variants cause progressive encephalopathy and infantile-onset refractory epilepsy.

Pathogenic variants in A Disintegrin And Metalloproteinase (ADAM) 22, the postsynaptic cell membrane receptor for the glycoprotein leucine-rich repeat glioma-inactivated protein 1 (LGI1), have been recently associated with recessive developmental and epileptic encephalopathy. However, so far, only two affected individuals have been described and many features of this disorder are unknown. We refine the phenotype and report 19 additional individuals harbouring compound heterozygous or homozygous inactivating ADAM22 variants, of whom 18 had clinical data available. Additionally, we provide follow-up data from two previously reported cases. All affected individuals exhibited infantile-onset, treatment-resistant epilepsy. Additional clinical features included moderate to profound global developmental delay/intellectual disability (20/20), hypotonia (12/20) and delayed motor development (19/20). Brain MRI findings included cerebral atrophy (13/20), supported by post-mortem histological examination in patient-derived brain tissue, cerebellar vermis atrophy (5/20), and callosal hypoplasia (4/20). Functional studies in transfected cell lines confirmed the deleteriousness of all identified variants and indicated at least three distinct pathological mechanisms: (i) defective cell membrane expression; (ii) impaired LGI1-binding; and/or (iii) impaired interaction with the postsynaptic density protein PSD-95. We reveal novel clinical and molecular hallmarks of ADAM22 deficiency and provide knowledge that might inform clinical management and early diagnostics.

De novo variants in ATP2B1 lead to neurodevelopmental delay.

Calcium (Ca2+) is a universal second messenger involved in synaptogenesis and cell survival; consequently, its regulation is important for neurons. ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) belongs to the family of ATP-driven calmodulin-dependent Ca2+ pumps that participate in the regulation of intracellular free Ca2+. Here, we clinically describe a cohort of 12 unrelated individuals with variants in ATP2B1 and an overlapping phenotype of mild to moderate global development delay. Additional common symptoms include autism, seizures, and distal limb abnormalities. Nine probands harbor missense variants, seven of which were in specific functional domains, and three individuals have nonsense variants. 3D structural protein modeling suggested that the variants have a destabilizing effect on the protein. We performed Ca2+ imaging after introducing all nine missense variants in transfected HEK293 cells and showed that all variants lead to a significant decrease in Ca2+ export capacity compared with the wild-type construct, thus proving their pathogenicity. Furthermore, we observed for the same variant set an incorrect intracellular localization of ATP2B1. The genetic findings and the overlapping phenotype of the probands as well as the functional analyses imply that de novo variants in ATP2B1 lead to a monogenic form of neurodevelopmental disorder.

De Novo SOX6 Variants Cause a Neurodevelopmental Syndrome Associated with ADHD, Craniosynostosis, and Osteochondromas.

SOX6 belongs to a family of 20 SRY-related HMG-box-containing (SOX) genes that encode transcription factors controlling cell fate and differentiation in many developmental and adult processes. For SOX6, these processes include, but are not limited to, neurogenesis and skeletogenesis. Variants in half of the SOX genes have been shown to cause severe developmental and adult syndromes, referred to as SOXopathies. We here provide evidence that SOX6 variants also cause a SOXopathy. Using clinical and genetic data, we identify 19 individuals harboring various types of SOX6 alterations and exhibiting developmental delay and/or intellectual disability; the individuals are from 17 unrelated families. Additional, inconstant features include attention-deficit/hyperactivity disorder (ADHD), autism, mild facial dysmorphism, craniosynostosis, and multiple osteochondromas. All variants are heterozygous. Fourteen are de novo, one is inherited from a mosaic father, and four offspring from two families have a paternally inherited variant. Intragenic microdeletions, balanced structural rearrangements, frameshifts, and nonsense variants are predicted to inactivate the SOX6 variant allele. Four missense variants occur in residues and protein regions highly conserved evolutionarily. These variants are not detected in the gnomAD control cohort, and the amino acid substitutions are predicted to be damaging. Two of these variants are located in the HMG domain and abolish SOX6 transcriptional activity in vitro. No clear genotype-phenotype correlations are found. Taken together, these findings concur that SOX6 haploinsufficiency leads to a neurodevelopmental SOXopathy that often includes ADHD and abnormal skeletal and other features.

Two novel cases expanding the phenotype of SETD2-related overgrowth syndrome.

The SETD2-related overgrowth syndrome is also called "Luscan-Lumish syndrome" (OMIM 616831) with the clinical characteristics of intellectual disability, speech delay, macrocephaly, facial dysmorphism, and autism spectrum disorders. We report on two novel patients a 4.5-year-old boy and a 23-year-old female adolescent with a speech and language developmental delay, autism spectrum disorder and macrocephaly, who were both diagnosed with SETD2-related overgrowth syndrome due to de novo frameshift mutations in the SETD2 gene. Features not previously described which were present in either one of our patients were nasal polyps, a large tongue with creases, a high pain threshold, constipation, and undescended testicles. These features may be related to the syndrome and may need special attention in future patients. Additionally, prevention of obesity should be an important point of attention for patients diagnosed with a SETD2-related overgrowth syndrome.

The Prognostic Value of AJCC Staging in Uveal Melanoma Is Enhanced by Adding Chromosome 3 and 8q Status.

Purpose: The American Joint Committee on Cancer (AJCC) staging system has been validated for use as a prognostic parameter in uveal melanoma (UM). We studied whether adding information regarding chromosome 3 and 8q status further enhances the prognostic value of this staging system. Methods: We retrospectively studied a cohort of 522 patients who had been treated for UM in two different centers between 1999 and 2015. The mean follow-up time was 47.7 months. Cumulative incidence curves were generated and regression analyses were performed for different combinations of AJCC staging and chromosome status. Death due to UM metastases was the primary endpoint. Results: In AJCC stage I cases, only patients with monosomy 3 as well as chromosome 8q gain died due to UM metastases (P < 0.001). Among patients with stage II and III tumors, those with monosomy 3 plus gain of chromosome 8q had the worst prognosis, whereas the clinical outcome of those with only one of these aberrations was intermediate (P < 0.001). Patients without monosomy 3 and 8q gain showed favorable prognosis, independent of their tumor's AJCC stage. In cases with monosomy 3, 8q gain, or both, adding AJCC stage improved the predictive value. Multivariable regression analyses demonstrated that AJCC staging and chromosome 3 and 8q status contain independent information about survival status. Conclusions: Combining information on AJCC staging and chromosome 3 and 8q status allows a more accurate prognostication in UM. We conclude that the prognostic value of the AJCC staging system can be improved by adding information regarding chromosome 3 and 8q status.

Mutations in the Chromatin Regulator Gene BRPF1 Cause Syndromic Intellectual Disability and Deficient Histone Acetylation.

Identification of over 500 epigenetic regulators in humans raises an interesting question regarding how chromatin dysregulation contributes to different diseases. Bromodomain and PHD finger-containing protein 1 (BRPF1) is a multivalent chromatin regulator possessing three histone-binding domains, one non-specific DNA-binding module, and several motifs for interacting with and activating three lysine acetyltransferases. Genetic analyses of fish brpf1 and mouse Brpf1 have uncovered an important role in skeletal, hematopoietic, and brain development, but it remains unclear how BRPF1 is linked to human development and disease. Here, we describe an intellectual disability disorder in ten individuals with inherited or de novo monoallelic BRPF1 mutations. Symptoms include infantile hypotonia, global developmental delay, intellectual disability, expressive language impairment, and facial dysmorphisms. Central nervous system and spinal abnormalities are also seen in some individuals. These clinical features overlap with but are not identical to those reported for persons with KAT6A or KAT6B mutations, suggesting that BRPF1 targets these two acetyltransferases and additional partners in humans. Functional assays showed that the resulting BRPF1 variants are pathogenic and impair acetylation of histone H3 at lysine 23, an abundant but poorly characterized epigenetic mark. We also found a similar deficiency in different lines of Brpf1-knockout mice. These data indicate that aberrations in the chromatin regulator gene BRPF1 cause histone H3 acetylation deficiency and a previously unrecognized intellectual disability syndrome.

Hypomorphic MKS1 mutation in a Pakistani family with mild Joubert syndrome and atypical features: Expanding the phenotypic spectrum of MKS1-related ciliopathies.

Postaxial polydactyly (PAP) is one of the most common congenital malformations observed in the general population. However, it can also occur as part of a syndrome. Unbiased genetic screening techniques such as exome sequencing are highly appropriate methods to provide a molecular diagnosis in patients with polydactyly due to the large number of mutated genes associated with it. The present study describes a consanguineous family of Pakistani origin with PAP, speech impairment, hearing impairment of variable degree, and proportionate short stature with no prominent intellectual disability or ophthalmological abnormalities. One affected individual of the family was subjected to exome sequencing which resulted in the identification of four homozygous variants including an in-frame deletion (c.1115_1117delCCT; p.(Ser372del) in MKS1, which was later shown to be the only variant segregating with the phenotype. In silico predictions supported the potential pathogenicity of the identified mutation. Additional clinical tests and MRI features of a patient in the family showed a molar tooth sign, which is a hallmark of Joubert syndrome. In conclusion, we have described a pathogenic variant in the MKS1 resulting in a mild Joubert syndrome phenotype, which broadens the spectrum of mutations in the MKS1. © 2016 Wiley Periodicals, Inc.

Parent-of-origin tumourigenesis is mediated by an essential imprinted modifier in SDHD-linked paragangliomas: SLC22A18 and CDKN1C are candidate tumour modifiers.

Mutations in SDHD and SDHAF2 (both located on chromosome 11) give rise to hereditary paraganglioma almost exclusively after paternal transmission of the mutation, and tumours often show loss of the entire maternal copy of chromosome 11. The 'Hensen' model postulates that a tumour modifier gene located on chromosome 11p15, a region known to harbour a cluster of imprinted genes, is essential to tumour formation. We observed decreased protein expression of the 11p15 candidate genes CDKN1C, SLC22A18 and ZNF215 evaluated in 60 SDHD-mutated tumours compared to normal carotid body tissue and non-SDH mutant tumours.We then created stable knockdown in vitro models, reasoning that the simultaneous knockdown of SDHD and a maternally expressed 11p15 modifier gene would enhance paraganglioma-related cellular characteristics compared to SDHD knockdown alone. Knockdown of SDHD in SNB19 and SHSY5Y cells resulted in the accumulation of succinate, the stabilization of HIF1 protein and a reduction in cell proliferation.Compared to single knockdown of SDHD, knockdown of SDHD together with SLC22A18 or with CDKN1C led to small but significant increases in cell proliferation and resistance to apoptosis, and to a gene expression profile closely related to the known transcriptional profile of SDH-deficient tumours. Of the 60 SDHD tumours investigated, four tumours showing retention of chromosome 11 showed SLC22A18 and CDKN1C expression levels comparable to levels in tumours showing loss of chromosome 11, suggesting loss of protein expression despite chromosomal retention.Our data strongly suggest that SLC22A18 and/or CDKN1C are tumour modifier genes involved in the tumourigenesis of SDHD-linked paraganglioma.

MECHANISMS IN ENDOCRINOLOGY: Novel genetic causes of short stature.

The fast technological development, particularly single nucleotide polymorphism array, array-comparative genomic hybridization, and whole exome sequencing, has led to the discovery of many novel genetic causes of growth failure. In this review we discuss a selection of these, according to a diagnostic classification centred on the epiphyseal growth plate. We successively discuss disorders in hormone signalling, paracrine factors, matrix molecules, intracellular pathways, and fundamental cellular processes, followed by chromosomal aberrations including copy number variants (CNVs) and imprinting disorders associated with short stature. Many novel causes of GH deficiency (GHD) as part of combined pituitary hormone deficiency have been uncovered. The most frequent genetic causes of isolated GHD are GH1 and GHRHR defects, but several novel causes have recently been found, such as GHSR, RNPC3, and IFT172 mutations. Besides well-defined causes of GH insensitivity (GHR, STAT5B, IGFALS, IGF1 defects), disorders of NFκB signalling, STAT3 and IGF2 have recently been discovered. Heterozygous IGF1R defects are a relatively frequent cause of prenatal and postnatal growth retardation. TRHA mutations cause a syndromic form of short stature with elevated T3/T4 ratio. Disorders of signalling of various paracrine factors (FGFs, BMPs, WNTs, PTHrP/IHH, and CNP/NPR2) or genetic defects affecting cartilage extracellular matrix usually cause disproportionate short stature. Heterozygous NPR2 or SHOX defects may be found in ∼3% of short children, and also rasopathies (e.g., Noonan syndrome) can be found in children without clear syndromic appearance. Numerous other syndromes associated with short stature are caused by genetic defects in fundamental cellular processes, chromosomal abnormalities, CNVs, and imprinting disorders.

Heterogeneity revealed by integrated genomic analysis uncovers a molecular switch in malignant uveal melanoma.

Gene expression profiles as well as genomic imbalances are correlated with disease progression in uveal melanoma (UM). We integrated expression and genomic profiles to obtain insight into the oncogenic mechanisms in development and progression of UM. We used tumor tissue from 64 enucleated eyes of UM patients for profiling. Mutations and genomic imbalances were quantified with digital PCR to study tumor heterogeneity and molecular pathogenesis. Gene expression analysis divided the UM panel into three classes. Class I presented tumors with a good prognosis and a distinct genomic make up that is characterized by 6p gain. The UM with a bad prognosis were subdivided into class IIa and class IIb. These classes presented similar survival risks but could be distinguished by tumor heterogeneity. Class IIa presented homogeneous tumors while class IIb tumors, on average, contained 30% of non-mutant cells. Tumor heterogeneity coincided with expression of a set of immune genes revealing an extensive immune infiltrate in class IIb tumors. Molecularly, class IIa and IIb presented the same genomic configuration and could only be distinguished by 8q copy number. Moreover, UM establish in the void of the immune privileged eye indicating that in IIb tumors the infiltrate is attracted by the UM. Combined our data show that chromosome 8q contains the locus that causes the immune phentotype of UM. UM thereby provides an unique opportunity to study immune attraction by tumors.

Heterozygous Loss-of-Function Mutations in DLL4 Cause Adams-Oliver Syndrome.

Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the presence of aplasia cutis congenita (ACC) of the scalp vertex and terminal limb-reduction defects. Cardiovascular anomalies are also frequently observed. Mutations in five genes have been identified as a cause for AOS prior to this report. Mutations in EOGT and DOCK6 cause autosomal-recessive AOS, whereas mutations in ARHGAP31, RBPJ, and NOTCH1 lead to autosomal-dominant AOS. Because RBPJ, NOTCH1, and EOGT are involved in NOTCH signaling, we hypothesized that mutations in other genes involved in this pathway might also be implicated in AOS pathogenesis. Using a candidate-gene-based approach, we prioritized DLL4, a critical NOTCH ligand, due to its essential role in vascular development in the context of cardiovascular features in AOS-affected individuals. Targeted resequencing of the DLL4 gene with a custom enrichment panel in 89 independent families resulted in the identification of seven mutations. A defect in DLL4 was also detected in two families via whole-exome or genome sequencing. In total, nine heterozygous mutations in DLL4 were identified, including two nonsense and seven missense variants, the latter encompassing four mutations that replace or create cysteine residues, which are most likely critical for maintaining structural integrity of the protein. Affected individuals with DLL4 mutations present with variable clinical expression with no emerging genotype-phenotype correlations. Our findings demonstrate that DLL4 mutations are an additional cause of autosomal-dominant AOS or isolated ACC and provide further evidence for a key role of NOTCH signaling in the etiology of this disorder.

Digital PCR validates 8q dosage as prognostic tool in uveal melanoma.

BACKGROUND: Uveal melanoma (UM) development and progression is correlated with specific molecular changes. Recurrent mutations in GNAQ and GNA11 initiate UM development while tumour progression is correlated with monosomy of chromosome 3 and gain of chromosome 8q. Hence, molecular analysis of UM is useful for diagnosis and prognosis. The aim of this study is to evaluate the use of digital PCR (dPCR) for molecular analysis of UM. METHODS: A series of 66 UM was analysed with dPCR for three hotspot mutations in GNAQ/GNA11 with mutation specific probes. The status of chromosomes 3 and 8 were analysed with genomic probes. The results of dPCR analysis were cross-validated with Sanger sequencing, SNP array analysis, and karyotyping. RESULTS: Using dPCR, we were able to reconstitute the molecular profile of 66 enucleated UM. With digital PCR, GNAQ/GNA11 mutations were detected in 60 of the 66 UM. Sanger sequencing revealed three rare variants, and, combined, these assays revealed GNAQ/GNA11 mutations in 95% of UM. Monosomy 3 was present in 43 and chromosome 8 aberrations in 52 of the 66 UM. Survival analysis showed that increasing 8q copy numbers were positively correlated with metastasis risk. CONCLUSION: Molecular analysis with dPCR is fast and sensitive. Just like the recurrent genomic aberrations of chromosome 3 and 8, hotspot mutations in GNAQ and GNA11 are effectively detected in heterogeneous samples. Increased sensitivity contributes to the number of mutations and chromosomal aberrations detected. Moreover, quantification of copy number with dPCR validated 8q dosage as a sensitive prognostic tool in UM, of which implementation in disease prediction models will further improve prognostication.

Prognostic parameters in uveal melanoma and their association with BAP1 expression.

AIM: To determine whether BAP1 gene and protein expression associates with different prognostic parameters in uveal melanoma and whether BAP1 expression correctly identifies patients as being at risk for metastases, following enucleation of the primary tumour. METHODS: Thirty cases of uveal melanoma obtained by enucleation between 1999 and 2004 were analysed for a variety of prognostic markers, including histological characteristics, chromosome aberrations obtained by fluorescence in situ hybridisation (FISH) and single nucleotide polymorphism (SNP) analysis and gene expression profiling. These parameters were compared with BAP1 gene expression and BAP1 immunostaining. RESULTS: The presence of monosomy of chromosome 3 as identified by the different chromosome 3 tests showed significantly increased HRs (FISH on isolated nuclei cut-off 30%: HR 11.6, p=0.002; SNP analysis: HR 20.3, p=0.004) for death due to metastasis. The gene expression profile class 2, based on the 15-gene expression profile, similarly provided a significantly increased HR for a poor outcome (HR 8.5, p=0.005). Lower BAP1 gene expression and negative BAP1 immunostaining (50% of 28 tumours were immunonegative) were both associated with these markers for prognostication: FISH cut-off 30% monosomy 3 (BAP1 gene expression: p=0.037; BAP1 immunostaining: p=0.001), SNP-monosomy 3 (BAP1 gene expression: p=0.008; BAP1 immunostaining: p=0.002) and class 2 profile (BAP1 gene expression: p<0.001; BAP1 immunostaining: p=0.001) and were themselves associated with an increased risk of death due to metastasis (BAP1 gene expression dichotomised: HR 8.7, p=0.006; BAP1 immunostaining: HR 4.0, p=0.010). CONCLUSIONS: Loss of BAP1 expression associated well with all of the methods currently used for prognostication and was itself predictive of death due to metastasis in uveal melanoma after enucleation, thereby emphasising the importance of further research on the role of BAP1 in uveal melanoma.

Different mutations in PDE4D associated with developmental disorders with mirror phenotypes.

BACKGROUND: Point mutations in PDE4D have been recently linked to acrodysostosis, an autosomal dominant disorder with skeletal dysplasia, severe brachydactyly, midfacial hypoplasia and intellectual disability. The purpose of the present study was to investigate clinical and cellular implications of different types of mutations in the PDE4D gene. METHODS: We studied five acrodysostosis patients and three patients with gene dose imbalances involving PDE4D clinically and by whole exome sequencing, Sanger sequencing and array comparative hybridisation. To evaluate the functional consequences of the PDE4D changes, we used overexpression of mutated human PDE4D message and morpholino-based suppression of pde4d in zebrafish. RESULTS: We identified three novel and two previously described PDE4D point mutations in the acrodysostosis patients and two deletions and one duplication involving PDE4D in three patients suffering from an intellectual disability syndrome with low body mass index, long fingers, toes and arms, prominent nose and small chin. When comparing symptoms in patients with missense mutations and gene dose imbalances involving PDE4D, a mirror phenotype was observed. By comparing overexpression of human mutated transcripts with pde4d knockdown in zebrafish embryos, we could successfully assay the pathogenicity of the mutations. CONCLUSIONS: Our findings indicate that haploinsufficiency of PDE4D results in a novel intellectual disability syndrome, the 5q12.1-haploinsufficiency syndrome, with several opposing features compared with acrodysostosis that is caused by dominant negative mutations. In addition, our results expand the spectrum of PDE4D mutations underlying acrodysostosis and indicate that, in contrast to previous reports, patients with PDE4D mutations may have significant hormone resistance with consequent endocrine abnormalities.

An activating mutation in the kinase homology domain of the natriuretic peptide receptor-2 causes extremely tall stature without skeletal deformities.

BACKGROUND: C-type natriuretic peptide (CNP)/natriuretic peptide receptor 2 (NPR2) signaling is essential for long bone growth. Enhanced CNP production caused by chromosomal translocations results in tall stature, a Marfanoid phenotype, and skeletal abnormalities. A similar phenotype was described in a family with an activating NPR2 mutation within the guanylyl cyclase domain. CASE: Here we describe an extremely tall male without skeletal deformities, with a novel NPR2 mutation (p.Arg655Cys) located in the kinase homology domain. OBJECTIVES: The objective of the study was to investigate the functional and structural effects of the NPR2 mutation. METHODS: Guanylyl cyclase activities of wild-type vs mutant NPR2 were analyzed in transfected human embryonic kidney 293 cells and in skin fibroblasts. The former were also used to study possible interactions between both isoforms. Homology modeling was performed to understand the molecular impact of the mutation. RESULTS: CNP-stimulated cGMP production by the mutant NPR2 was markedly increased in patient skin fibroblasts and transfected human embryonic kidney 293 cells. The stimulatory effects of ATP on CNP-dependent guanylyl cyclase activity were augmented, suggesting that this novel mutation enhances both the responsiveness of NPR2 to CNP and its allosteric modulation/stabilization by ATP. Coimmunoprecipitation showed that wild-type and mutant NPR2 can form stable heterodimers, suggesting a dominant-positive effect. In accordance with augmented endogenous receptor activity, plasma N-terminal pro-CNP (a marker of CNP production in tissues) was reduced in the proband. CONCLUSIONS: We report the first activating mutation within the kinase homology domain of NPR2, resulting in extremely tall stature. Our observations emphasize the important role of this domain in the regulation of guanylyl cyclase activity and bone growth in response to CNP.