Compound heterozygous KCTD7 variants in progressive myoclonus epilepsy.

KCTD7 is a member of the potassium channel tetramerization domain-containing protein family and has been associated with progressive myoclonic epilepsy (PME), characterized by myoclonus, epilepsy, and neurological deterioration. Here we report four affected individuals from two unrelated families in which we identified KCTD7 compound heterozygous single nucleotide variants through exome sequencing. RNAseq was used to detect a non-annotated splicing junction created by a synonymous variant in the second family. Whole-cell patch-clamp analysis of neuroblastoma cells overexpressing the patients' variant alleles demonstrated aberrant potassium regulation. While all four patients experienced many of the common clinical features of PME, they also showed variable phenotypes not previously reported, including dysautonomia, brain pathology findings including a significantly reduced thalamus, and the lack of myoclonic seizures. To gain further insight into the pathogenesis of the disorder, zinc finger nucleases were used to generate kctd7 knockout zebrafish. Kctd7 homozygous mutants showed global dysregulation of gene expression and increased transcription of c-fos, which has previously been correlated with seizure activity in animal models. Together these findings expand the known phenotypic spectrum of KCTD7-associated PME, report a new animal model for future studies, and contribute valuable insights into the disease.

Expanding the phenotype of COPA syndrome: a kindred with typical and atypical features.

BACKGROUND: Copa syndrome is a rare autosomal dominant disorder with abnormal intracellular vesicle trafficking. The objective of this work is to expand the knowledge about this disorder by delineating phenotypic features of an unreported COPA family. METHODS AND RESULTS: A heterozygous missense variant (c.698 G>A, p.Arg233His) in COPA was identified in four members of a three-generation kindred with lung, autoimmune and malignant disease of unknown aetiology. Ages of onset were 56, 26, 16 and 1 year, with earlier age of onset in successive generations. Presenting symptoms were cough and dyspnoea. Findings included small lung cysts, follicular bronchiolitis, interstitial lung disease, neuroendocrine cell hyperplasia, rheumatoid arthritis, avascular necrosis and select abnormal autoimmune serologies. Neither alveolar haemorrhage nor glomerular disease were present. Features not previously associated with Copa syndrome included neuromyelitis optica, pulmonary carcinoid tumour, clear cell renal carcinoma, renal cysts, hepatic cysts, nephrolithiasis, pyelonephritis and meningitis. Longitudinal evaluations demonstrated slow progression of lung disease and extrapulmonary cysts. CONCLUSIONS: Worsening severity with successive generations may be observed in Copa syndrome. Extrapulmonary cysts, malignancies, autoimmune neurological disorders and infections are clinical features that may be associated with Copa syndrome. Further studies are indicated to fully define the phenotypic spectrum of this disorder.

Combined alpha-delta platelet storage pool deficiency is associated with mutations in GFI1B.

Combined alpha-delta platelet storage pool deficiency is characterized by the absence or reduction in the number of both alpha granules and dense bodies. This disorder can have variable severity as well as a variable inheritance pattern. We describe two patients from unrelated families with combined alpha-delta storage pool deficiency due to mutations in GFI1B, a zinc finger protein known to act as a transcriptional repressor of various genes. We demonstrate that this disease is associated with either a heterozygous mutation (de novo or familial) abrogating the binding of the zinc fingers with the promoter of its target genes, or by hypomorphic biallelic mutations in GFI1B leading to autosomal recessive inheritance.

ATP1A3 Mutation in Adult Rapid-Onset Ataxia.

A 21-year old male presented with ataxia and dysarthria that had appeared over a period of months. Exome sequencing identified a de novo missense variant in ATP1A3, the gene encoding the α3 subunit of Na,K-ATPase. Several lines of evidence suggest that the variant is causative. ATP1A3 mutations can cause rapid-onset dystonia-parkinsonism (RDP) with a similar age and speed of onset, as well as severe diseases of infancy. The patient's ATP1A3 p.Gly316Ser mutation was validated in the laboratory by the impaired ability of the expressed protein to support the growth of cultured cells. In a crystal structure of Na,K-ATPase, the mutated amino acid was directly apposed to a different amino acid mutated in RDP. Clinical evaluation showed that the patient had many characteristics of RDP, however he had minimal fixed dystonia, a defining symptom of RDP. Successive magnetic resonance imaging (MRI) revealed progressive cerebellar atrophy, explaining the ataxia. The absence of dystonia in the presence of other RDP symptoms corroborates other evidence that the cerebellum contributes importantly to dystonia pathophysiology. We discuss the possibility that a second de novo variant, in ubiquilin 4 (UBQLN4), a ubiquitin pathway component, contributed to the cerebellar neurodegenerative phenotype and differentiated the disease from other manifestations of ATP1A3 mutations. We also show that a homozygous variant in GPRIN1 (G protein-regulated inducer of neurite outgrowth 1) deletes a motif with multiple copies and is unlikely to be causative.

Hereditary Benign Intraepithelial Dyskeratosis: Report of a Case and Re-examination of the Evidence for Locus Heterogeneity.

BACKGROUND: Hereditary benign intraepithelial dyskeratosis (HBID) is a rare autosomal-dominant disorder of the conjunctiva and oral mucosa first described in and predominantly affecting descendents of Haliwa-Saponi Native Americans. We report a spontaneous case of histopathologically-confirmed HBID affecting an individual not of Native American ancestry. MATERIALS AND METHODS: Report of a case with histopathologic examination of an excised conjunctival specimen as well as molecular and cytogenetic analysis. RESULTS: A Caucasian boy with a history of oral lesions and conjunctival injection from birth developed bilateral corneal opacities at age 5 and underwent penetrating keratoplasty, with recurrence of the corneal opacification shortly after surgery. Examination of a conjunctival biopsy specimen revealed features consistent with HBID. Copy number variant (CNV) analysis revealed a de novo 4q35 duplication that overlapped the duplication previously associated with HBID, although no genes were identified in the common interval. NLRP1 gene sequencing failed to reveal a presumed pathogenic variant. CONCLUSIONS: HBID may develop de novo in individuals who are not of Native American ancestry. The absence of coding regions in a duplicated region of 4q35 common to both the individual that we report and previously associated with HBID raises questions regarding the significance of this CNV in the pathogenesis of HBID.

MED23-associated intellectual disability in a non-consanguineous family.

Intellectual disability (ID) is a heterogeneous condition arising from a variety of environmental and genetic factors. Among these causes are defects in transcriptional regulators. Herein, we report on two brothers in a nonconsanguineous family with novel compound heterozygous, disease-segregating mutations (NM_015979.3: [3656A > G];[4006C > T], NP_057063.2: [H1219R];[R1336X]) in MED23. This gene encodes a subunit of the Mediator complex that modulates the expression of RNA polymerase II-dependent genes. These brothers, who had profound ID, spasticity, congenital heart disease, brain abnormalities, and atypical electroencephalography, represent the first case of MED23-associated ID in a non-consanguineous family. They also expand upon the clinical features previously reported for mutations in this gene.

Impaired osteoblast and osteoclast function characterize the osteoporosis of Snyder - Robinson syndrome.

BACKGROUND: Snyder-Robinson Syndrome (SRS) is an X-linked intellectual disability disorder also characterized by osteoporosis, scoliosis, and dysmorphic facial features. It is caused by mutations in SMS, a ubiquitously expressed gene encoding the polyamine biosynthetic enzyme spermine synthase. We hypothesized that the tissue specificity of SRS arises from differential sensitivity to spermidine toxicity or spermine deficiency. METHODS: We performed detailed clinical, endocrine, histopathologic, and morphometric studies on two affected brothers with a spermine synthase loss of function mutation (NM_004595.4:c.443A > G, p.Gln148Arg). We also measured spermine and spermidine levels in cultured human bone marrow stromal cells (hBMSCs) and fibroblasts using the Biochrom 30 polyamine protocol and assessed the osteogenic potential of hBMSCs. RESULTS: In addition to the known tissue-specific features of SRS, the propositi manifested retinal pigmentary changes, recurrent episodes of hyper- and hypoglycemia, nephrocalcinosis, renal cysts, and frequent respiratory infections. Bone histopathology and morphometry identified a profound depletion of osteoblasts and osteoclasts, absence of a trabecular meshwork, a low bone volume and a thin cortex. Comparison of cultured fibroblasts from affected and unaffected individuals showed relatively small changes in polyamine content, whereas comparison of cultured osteoblasts identified marked differences in spermidine and spermine content. Osteogenic differentiation of the SRS-derived hBMSCs identified a severe deficiency of calcium phosphate mineralization. CONCLUSIONS: Our findings support the hypothesis that cell specific alterations in polyamine metabolism contribute to the tissue specificity of SRS features, and that the low bone density arises from a failure of mineralization.

Circulating fibrocytes as biomarker of prognosis in Hermansky-Pudlak syndrome.

RATIONALE: The rate of progression of most interstitial lung diseases (ILD) is unpredictable. Fibrocytes are circulating bone marrow-derived cells that have been implicated in the pathogenesis of lung fibrosis. Hermansky-Pudlak syndrome (HPS), a genetic cause of ILD in early adulthood, allows for study of biomarkers of ILD in a homogeneous population at near-certain risk of developing fibrotic lung disease. OBJECTIVES: To test the hypothesis that, in subjects with HPS, the number or phenotype of circulating fibrocytes predicts progression and outcome of ILD. METHODS: We measured circulating fibrocyte counts and chemokine levels in a cohort of subjects with HPS and healthy control subjects and correlated the results to disease outcome. MEASUREMENTS AND MAIN RESULTS: In a cross-sectional analysis, peripheral blood fibrocyte concentrations were markedly elevated in a subset of subjects with HPS who had ILD but not subjects without lung disease or normal control subjects. The blood concentration of fibrocytes expressing the chemokine receptor CXCR4 correlated significantly with the plasma concentration of the CXCR4 ligand, CXCL12. In a longitudinal study, we found marked episodic elevations in circulating fibrocyte counts over a median follow-up period of 614 days. Elevations in both maximal values and final values of peripheral blood CXCR4(+) fibrocyte concentration were strongly associated with death from ILD. CONCLUSIONS: CXCR4(+) fibrocyte concentration may be useful as a biomarker for outcome of ILD in subjects with HPS.

Diminutive somatic deletions in the 5q region lead to a phenotype atypical of classical 5q- syndrome.

Classical 5q- syndrome is an acquired macrocytic anemia of the elderly. Similar to Diamond Blackfan anemia (DBA), an inherited red cell aplasia, the bone marrow is characterized by a paucity of erythroid precursors. RPS14 deletions in combination with other deletions in the region have been implicated as causative of the 5q- syndrome phenotype. We asked whether smaller, less easily detectable deletions could account for a syndrome with a modified phenotype. We employed single-nucleotide polymorphism array genotyping to identify small deletions in patients diagnosed with DBA and other anemias lacking molecular diagnoses. Diminutive mosaic deletions involving RPS14 were identified in a 5-year-old patient with nonclassical DBA and in a 17-year-old patient with myelodysplastic syndrome. Patients with nonclassical DBA and other hypoproliferative anemias may have somatically acquired 5q deletions with RPS14 haploinsufficiency not identified by fluorescence in situ hybridization or cytogenetic testing, thus refining the spectrum of disorders with 5q- deletions.

The National Institutes of Health Undiagnosed Diseases Program: insights into rare diseases.

PURPOSE: This report describes the National Institutes of Health Undiagnosed Diseases Program, details the Program's application of genomic technology to establish diagnoses, and details the Program's success rate during its first 2 years. METHODS: Each accepted study participant was extensively phenotyped. A subset of participants and selected family members (29 patients and 78 unaffected family members) was subjected to an integrated set of genomic analyses including high-density single-nucleotide polymorphism arrays and whole exome or genome analysis. RESULTS: Of 1,191 medical records reviewed, 326 patients were accepted and 160 were admitted directly to the National Institutes of Health Clinical Center on the Undiagnosed Diseases Program service. Of those, 47% were children, 55% were females, and 53% had neurologic disorders. Diagnoses were reached on 39 participants (24%) on clinical, biochemical, pathologic, or molecular grounds; 21 diagnoses involved rare or ultra-rare diseases. Three disorders were diagnosed based on single-nucleotide polymorphism array analysis and three others using whole exome sequencing and filtering of variants. Two new disorders were discovered. Analysis of the single-nucleotide polymorphism array study cohort revealed that large stretches of homozygosity were more common in affected participants relative to controls. CONCLUSION: The National Institutes of Health Undiagnosed Diseases Program addresses an unmet need, i.e., the diagnosis of patients with complex, multisystem disorders. It may serve as a model for the clinical application of emerging genomic technologies and is providing insights into the characteristics of diseases that remain undiagnosed after extensive clinical workup.

Ribosomal protein gene deletions in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital BM failure syndrome characterized by hypoproliferative anemia, associated physical abnormalities, and a predisposition to cancer. Perturbations of the ribosome appear to be critically important in DBA; alterations in 9 different ribosomal protein genes have been identified in multiple unrelated families, along with rarer abnormalities of additional ribosomal proteins. However, at present, only 50% to 60% of patients have an identifiable genetic lesion by ribosomal protein gene sequencing. Using genome-wide single-nucleotide polymorphism array to evaluate for regions of recurrent copy variation, we identified deletions at known DBA-related ribosomal protein gene loci in 17% (9 of 51) of patients without an identifiable mutation, including RPS19, RPS17, RPS26, and RPL35A. No recurrent regions of copy variation at novel loci were identified. Because RPS17 is a duplicated gene with 4 copies in a diploid genome, we demonstrate haploinsufficient RPS17 expression and a small subunit ribosomal RNA processing abnormality in patients harboring RPS17 deletions. Finally, we report the novel identification of variable mosaic loss involving known DBA gene regions in 3 patients from 2 kindreds. These data suggest that ribosomal protein gene deletion is more common than previously suspected and should be considered a component of the initial genetic evaluation in cases of suspected DBA.

Homozygosity mapping and whole-exome sequencing to detect SLC45A2 and G6PC3 mutations in a single patient with oculocutaneous albinism and neutropenia.

We evaluated a 32-year-old woman whose oculocutaneous albinism (OCA), bleeding diathesis, neutropenia, and history of recurrent infections prompted consideration of the diagnosis of Hermansky-Pudlak syndrome type 2. This was ruled out because of the presence of platelet δ-granules and absence of AP3B1 mutations. As parental consanguinity suggested an autosomal recessive mode of inheritance, we employed homozygosity mapping, followed by whole-exome sequencing, to identify two candidate disease-causing genes, SLC45A2 and G6PC3. Conventional dideoxy sequencing confirmed pathogenic mutations in SLC45A2, associated with OCA type 4 (OCA-4), and G6PC3, associated with neutropenia. The substantial reduction of SLC45A2 protein in the patient's melanocytes caused the mislocalization of tyrosinase from melanosomes to the plasma membrane and also led to the incorporation of tyrosinase into exosomes and secretion into the culture medium, explaining the hypopigmentation in OCA-4. Our patient's G6PC3 mRNA expression level was also reduced, leading to increased apoptosis of her fibroblasts under endoplasmic reticulum stress. To our knowledge, this report describes the first North American patient with OCA-4, the first culture of human OCA-4 melanocytes, and the use of homozygosity mapping, followed by whole-exome sequencing, to identify disease-causing mutations in multiple genes in a single affected individual.

PTPRF is disrupted in a patient with syndromic amastia.

BACKGROUND: The presence of mammary glands distinguishes mammals from other organisms. Despite significant advances in defining the signaling pathways responsible for mammary gland development in mice, our understanding of human mammary gland development remains rudimentary. Here, we identified a woman with bilateral amastia, ectodermal dysplasia and unilateral renal agenesis. She was found to have a chromosomal balanced translocation, 46,XX,t(1;20)(p34.1;q13.13). In addition to characterization of her clinical and cytogenetic features, we successfully identified the interrupted gene and studied its consequences. METHODS: Characterization of the breakpoints was performed by molecular cytogenetic techniques. The interrupted gene was further analyzed using quantitative real-time PCR and western blotting. Mutation analysis and high-density SNP array were carried out in order to find a pathogenic mutation. Allele segregations were obtained by haplotype analysis. RESULTS: We enabled to identify its breakpoint on chromosome 1 interrupting the protein tyrosine receptor type F gene (PTPRF). While the patient's mother and sisters also harbored the translocated chromosome, their non-translocated chromosomes 1 were different from that of the patient. Although a definite pathogenic mutation on the paternal allele could not be identified, PTPRF's RNA and protein of the patient were significantly less than those of her unaffected family members. CONCLUSIONS: Although ptprf has been shown to involve in murine mammary gland development, no evidence has incorporated PTPRF in human organ development. We, for the first time, demonstrated the possible association of PTPRF with syndromic amastia, making it a prime candidate to investigate for its spatial and temporal roles in human breast development.

Vascular pathology of medial arterial calcifications in NT5E deficiency: implications for the role of adenosine in pseudoxanthoma elasticum.

Arterial Calcification due to Deficiency of CD73 (ACDC) results from mutations in the NT5E gene encoding the 5' exonucleotidase, CD73. We now describe the third familial case of ACDC, including radiological and histopathological details of the arterial calcifications. The medial lesions involve the entire circumference of the elastic lamina, in contrast to the intimal plaque-like disease of atherosclerosis. The demonstration of broken and fragmented elastic fibers leading to generalized vascular calcification suggests an analogy to pseudoxanthoma elasticum (PXE), which exhibits similar histopathology. Classical PXE is caused by deficiency of ABCC6, a C type ABC transporter whose ligand is unknown. Other C type ABC proteins transport nucleotides, so the newly described role of adenosine in inhibiting vascular calcification, along with the similarity of ACDC and PXE with respect to vascular pathology, suggests that adenosine may be the ligand for ABCC6.

NT5E mutations and arterial calcifications.

BACKGROUND: Arterial calcifications are associated with increased cardiovascular risk, but the genetic basis of this association is unclear. METHODS: We performed clinical, radiographic, and genetic studies in three families with symptomatic arterial calcifications. Single-nucleotide-polymorphism analysis, targeted gene sequencing, quantitative polymerase-chain-reaction assays, Western blotting, enzyme measurements, transduction rescue experiments, and in vitro calcification assays were performed. RESULTS: We identified nine persons with calcifications of the lower-extremity arteries and hand and foot joint capsules: all five siblings in one family, three siblings in another, and one patient in a third family. Serum calcium, phosphate, and vitamin D levels were normal. Affected members of Family 1 shared a single 22.4-Mb region of homozygosity on chromosome 6 and had a homozygous nonsense mutation (c.662C→A, p.S221X) in NT5E, encoding CD73, which converts AMP to adenosine. Affected members of Family 2 had a homozygous missense mutation (c.1073G→A, p.C358Y) in NT5E. The proband of Family 3 was a compound heterozygote for c.662C→A and c.1609dupA (p.V537fsX7). All mutations found in the three families result in nonfunctional CD73. Cultured fibroblasts from affected members of Family 1 showed markedly reduced expression of NT5E messenger RNA, CD73 protein, and enzyme activity, as well as increased alkaline phosphatase levels and accumulated calcium phosphate crystals. Genetic rescue experiments normalized the CD73 and alkaline phosphatase activity in patients' cells, and adenosine treatment reduced the levels of alkaline phosphatase and calcification. CONCLUSIONS: We identified mutations in NT5E in members of three families with symptomatic arterial and joint calcifications. This gene encodes CD73, which converts AMP to adenosine, supporting a role for this metabolic pathway in inhibiting ectopic tissue calcification. (Funded by the National Human Genome Research Institute and the National Heart, Lung, and Blood Institute of the National Institutes of Health.).

Novel 47.5-kb deletion in RAB27A results in severe Griscelli Syndrome Type 2.

Griscelli syndrome (GS), a rare autosomal recessive disorder characterized by partial albinism and immunological impairment and/or severe neurological impairment, results from mutations in the MYO5A (GS1), RAB27A (GS2), or MLPH (GS3) genes. We identified a Hispanic patient born of a consanguineous union who presented with immunodeficiency, partial albinism, hepatic dysfunction, hemophagocytosis, neurological impairment, nystagmus, and silvery hair indicative of Griscelli Syndrome Type 2 (GS2). We screened for point mutations, but only exons 2-6 of the patient's DNA could be PCR-amplified. Whole genome analysis using the Illumina 1M-Duo DNA Analysis BeadChip identified a homozygous deletion in the patient's DNA. The exact breakpoints of the 47.5-kb deletion were identified as chr15q15-q21.1: g.53332432_53379990del (NCBI Build 37.1); the patient lacks the promoter and 5'UTR regions of RAB27A, thus confirming the diagnosis of GS2.

Chediak-Higashi syndrome with early developmental delay resulting from paternal heterodisomy of chromosome 1.

Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disease characterized by variable oculocutaneous albinism, immunodeficiency, mild bleeding diathesis, and an accelerated lymphoproliferative state. Abnormal lysosome-related organelle membrane function leads to the accumulation of large intracellular vesicles in several cell types, including granulocytes, melanocytes, and platelets. This report describes a severe case of CHS resulting from paternal heterodisomy of chromosome 1, causing homozygosity for the most distal nonsense mutation (p.E3668X, exon 50) reported to date in the LYST/CHS1 gene. The mutation is located in the WD40 region of the CHS1 protein. The patient's fibroblasts expressed no detectable CHS1. Besides manifesting the classical CHS findings, the patient exhibited hypotonia and global developmental delays, raising concerns about other effects of heterodisomy. An interstitial 747 kb duplication on 6q14.2-6q14.3 was identified in the propositus and paternal samples by comparative genomic hybridization. SNP genotyping revealed no additional whole chromosome or segmental isodisomic regions or other dosage variations near the crossover breakpoints on chromosome 1. Unmasking of a separate autosomal recessive cause of developmental delay, or an additive effect of the paternal heterodisomy, could underlie the severity of the phenotype in this patient.

Alveolar macrophage dysregulation in Hermansky-Pudlak syndrome type 1.

RATIONALE: Individuals with Hermansky-Pudlak syndrome type 1 (HPS-1), an autosomal recessive disorder characterized by defective biogenesis of lysosome-related organelles, develop an accelerated form of progressive fibrotic lung disease. The etiology of pulmonary fibrosis associated with HPS-1 is unknown. OBJECTIVES: To investigate the potential pathogenesis of pulmonary fibrosis in HPS-1, lung cells and proteins from individuals with HPS-1 were studied. METHODS: Forty-one subjects with HPS-1 with and without pulmonary fibrosis were evaluated with pulmonary function tests, high-resolution computed tomography scan, and bronchoscopy. Bronchoalveolar lavage cells and analytes were analyzed. MEASUREMENTS AND MAIN RESULTS: Concentrations of total bronchoalveolar lavage cells and alveolar macrophages were significantly higher in epithelial lining fluid from subjects with HPS-1 with and without pulmonary fibrosis compared with healthy research volunteers. Concentrations of cytokines and chemokines (i.e., monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and granulocyte-macrophage colony-stimulating factor) in alveolar epithelial lining fluid were significantly higher in subjects with HPS-1 with and without pulmonary fibrosis compared with healthy research volunteers (P < 0.001). In vitro, HPS-1 pulmonary fibrosis alveolar macrophages, which did not express HPS1 mRNA, secreted significantly higher concentrations of monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and regulated upon activation, normal T cell expressed and secreted (RANTES) protein compared with normal cells (P = 0.001, P = 0.014, and P = 0.011, respectively). Pirfenidone suppressed HPS-1 alveolar macrophage cytokine and chemokine secretion in vitro in a dose-dependent manner. CONCLUSIONS: In HPS-1, alveolar inflammation predominantly involves macrophages and is associated with high lung concentrations of cytokines and chemokines. HPS-1 alveolar macrophages provide a model system in which to study the pathogenesis and treatment of HPS pulmonary fibrosis.