Richard E. Person

Molecular Findings Among Patients Referred for Clinical Whole-Exome Sequencing

IMPORTANCE Clinical whole-exome sequencing is increasingly used for diagnostic evaluation of patients with suspected genetic disorders. OBJECTIVE To perform clinical whole-exome sequencing and report (1) the rate of molecular diagnosis among phenotypic groups, (2) the spectrum of genetic alterations contributing to disease, and (3) the prevalence of medically actionable incidental findings such as FBN1 mutations causing Marfan syndrome. DESIGN, SETTING, AND PATIENTS Observational study of 2000 consecutive patients with clinical whole-exome sequencing analyzed between June 2012 and August 2014. Whole-exome sequencing tests were performed at a clinical genetics laboratory in the United States. Results were reported by clinical molecular geneticists certified by the American Board of Medical Genetics and Genomics. Tests were ordered by the patients physician. The patients were primarily pediatric (1756 [88%]; mean age, 6 years; 888 females [44%], 1101 males [55%], and 11 fetuses [1% gender unknown]), demonstrating diverse clinical manifestations most often including nervous system dysfunction such as developmental delay. MAIN OUTCOMES AND MEASURES Whole-exome sequencing diagnosis rate overall and by phenotypic category, mode of inheritance, spectrum of genetic events, and reporting of incidental findings. RESULTS A molecular diagnosis was reported for 504 patients (25.2%) with 58% of the diagnostic mutations not previously reported. Molecular diagnosis rates for each phenotypic category were 143/526 (27.2%; 95% CI, 23.5%-31.2%) for the neurological group, 282/1147 (24.6%; 95% CI, 22.1%-27.2%) for the neurological plus other organ systems group, 30/83 (36.1%; 95% CI, 26.1%-47.5%) for the specific neurological group, and 49/244 (20.1%; 95% CI, 15.6%-25.8%) for the nonneurological group. The Mendelian disease patterns of the 527 molecular diagnoses included 280 (53.1%) autosomal dominant, 181 (34.3%) autosomal recessive (including 5 with uniparental disomy), 65 (12.3%) X-linked, and 1 (0.2%) mitochondrial. Of 504 patients with a molecular diagnosis, 23 (4.6%) had blended phenotypes resulting from 2 single gene defects. About 30% of the positive cases harbored mutations in disease genes reported since 2011. There were 95 medically actionable incidental findings in genes unrelated to the phenotype but with immediate implications for management in 92 patients (4.6%), including 59 patients (3%) with mutations in genes recommended for reporting by the American College of Medical Genetics and Genomics. CONCLUSIONS AND RELEVANCE Whole-exome sequencing provided a potential molecular diagnosis for 25% of a large cohort of patients referred for evaluation of suspected genetic conditions, including detection of rare genetic events and new mutations contributing to disease. The yield of whole-exome sequencing may offer advantages over traditional molecular diagnostic approaches in certain patients.

Prader-Willi phenotype caused by paternal deficiency for the HBII-85 C/D box small nucleolar RNA cluster.

Prader-Willi syndrome (PWS) is caused by deficiency for one or more paternally expressed imprinted transcripts within chromosome 15q11-q13, including SNURF-SNRPN and multiple small nucleolar RNAs (snoRNAs). Balanced chromosomal translocations that preserve expression of SNURF-SNRPN and centromeric genes but separate the snoRNA HBII-85 cluster from its promoter cause PWS. A microdeletion of the HBII-85 snoRNAs in a child with PWS provides, in combination with previous data, effectively conclusive evidence that deficiency of HBII-85 snoRNAs causes the key characteristics of the PWS phenotype, although some atypical features suggest that other genes in the region may make more subtle phenotypic contributions.

Mutations in ELA2 , encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis

Human cyclic haematopoiesis (cyclic neutropenia, MIM 162800) is an autosomal dominant disease in which blood-cell production from the bone marrow oscillates with 21-day periodicity. Circulating neutrophils vary between almost normal numbers and zero. During intervals of neutropenia, affected individuals are at risk for opportunistic infection. Monocytes, platelets, lymphocytes and reticulocytes also cycle with the same frequency. Here we use a genome-wide screen and positional cloning to map the locus to chromosome 19p13.3. We identified 7 different single-base substitutions in the gene (ELA2) encoding neutrophil elastase (EC 3.4.21.37, also known as leukocyte elastase, elastase 2 and medullasin), a serine protease of neutrophil and monocyte granules, on unique haplotypes in 13 of 13 families as well as a new mutation in a sporadic case. Neutrophil elastase (a 240-aa mature protein predominantly found in neutrophil granules) is the target for protease inhibition by α1-antitrypsin, and its unopposed release destroys tissue at sites of inflammation. We hypothesize that a perturbed interaction between neutrophil elastase and serpins or other substrates may regulate mechanisms governing the clock-like timing of haematopoiesis.

Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2

Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic. We therefore screened GFI1 as a candidate for association with neutropenia in affected individuals without mutations in ELA2 (encoding neutrophil elastase), the most common cause of severe congenital neutropenia (SCN; ref. 3). We found dominant negative zinc finger mutations that disable transcriptional repressor activity. The phenotype also includes immunodeficient lymphocytes and production of a circulating population of myeloid cells that appear immature. We show by chromatin immunoprecipitation, gel shift, reporter assays and elevated expression of ELA2 in vivo in neutropenic individuals that GFI1 represses ELA2, linking these two genes in a common pathway involved in myeloid differentiation.

Paternally inherited microdeletion at 15q11.2 confirms a significant role for the SNORD116 C/D box snoRNA cluster in Prader–Willi syndrome

Prader–Willi syndrome (PWS) is a neurobehavioral disorder manifested by infantile hypotonia and feeding difficulties in infancy, followed by morbid obesity secondary to hyperphagia. It is caused by deficiency of paternally expressed transcript(s) within the human chromosome region 15q11.2. PWS patients harboring balanced chromosomal translocations with breakpoints within small nuclear ribonucleoprotein polypeptide N (SNRPN) have provided indirect evidence for a role for the imprinted C/D box containing small nucleolar RNA (snoRNA) genes encoded downstream of SNRPN. In addition, recently published data provide strong evidence in support of a role for the snoRNA SNORD116 cluster (HBII-85) in PWS etiology. In this study, we performed detailed phenotypic, cytogenetic, and molecular analyses including chromosome analysis, array comparative genomic hybridization (array CGH), expression studies, and single-nucleotide polymorphism (SNP) genotyping for parent-of-origin determination of the 15q11.2 microdeletion on an 11-year-old child expressing the major components of the PWS phenotype. This child had an ∼236.29 kb microdeletion at 15q11.2 within the larger Prader–Willi/Angelman syndrome critical region that included the SNORD116 cluster of snoRNAs. Analysis of SNP genotypes in proband and mother provided evidence in support of the deletion being on the paternal chromosome 15. This child also met most of the major PWS diagnostic criteria including infantile hypotonia, early-onset morbid obesity, and hypogonadism. Identification and characterization of this case provide unequivocal evidence for a critical role for the SNORD116 snoRNA molecules in PWS pathogenesis. Array CGH testing for genomic copy-number changes in cases with complex phenotypes is proving to be invaluable in detecting novel alterations and enabling better genotype–phenotype correlations.

Mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase

Cyclic hematopoiesis is a stem cell disease in which the number of neutrophils and other blood cells oscillates in weekly phases. Autosomal dominant mutations of ELA2, encoding the protease neutrophil elastase, found in lysosome-like granules, cause cyclic hematopoiesis and most cases of the pre-leukemic disorder severe congenital neutropenia (SCN; ref. 3) in humans. Over 20 different mutations of neutrophil elastase have been identified, but their consequences are elusive, because they confer no consistent effects on enzymatic activity. The similar autosomal recessive disease of dogs, canine cyclic hematopoiesis, is not caused by mutations in ELA2 (data not shown). Here we show that homozygous mutation of the gene encoding the dog adaptor protein complex 3 (AP3) β-subunit, directing trans-Golgi export of transmembrane cargo proteins to lysosomes, causes canine cyclic hematopoiesis. C-terminal processing of neutrophil elastase exposes an AP3 interaction signal responsible for redirecting neutrophil elastase trafficking from membranes to granules. Disruption of either neutrophil elastase or AP3 perturbs the intracellular trafficking of neutrophil elastase. Most mutations in ELA2 that cause human cyclic hematopoiesis prevent membrane localization of neutrophil elastase, whereas most mutations in ELA2 that cause SCN lead to exclusive membrane localization.

Ube3a-ATS is an atypical RNA polymerase II transcript that represses the paternal expression of Ube3a

The Angelman syndrome gene, UBE3A, is subject to genomic imprinting controlled by mechanisms that are only partially understood. Its antisense transcript, UBE3A-ATS, is also imprinted and hypothesized to suppress UBE3A in cis. In this research, we showed that the mouse antisense ortholog, Ube3a-ATS, was transcribed by RNA polymerase (RNAP) II. However, unlike typical protein-coding transcripts, Ube3a-ATS was not poly-adenylated and was localized exclusively in the nucleus. It was relatively unstable with a half-life of 4 h, shorter than most protein-coding RNAs tested. To understand the role of Ube3a-ATS in vivo, a mouse model with a 0.9-kb genomic deletion over the paternal Snrpn major promoter was studied. The mice showed partial activation of paternal Ube3a, with decreased expression of Ube3a-ATS but not any imprinting defects in the Prader-Willi syndrome/Angelman syndrome region. A novel cell culture model was also generated with a transcriptional termination cassette inserted downstream of Ube3a on the paternal chromosome to reduce Ube3a-ATS transcription. In neuronally differentiated embryonic stem (ES) cells, paternal Ube3a was found to be expressed at a high level, comparable with that of the maternal allele. To further characterize the antisense RNA, a strand-specific microarray was performed. Ube3a-ATS was detectable across the entire locus of Ube3a and extended beyond the transcriptional start site of Ube3a. In summary, we conclude that Ube3a-ATS is an atypical RNAPII transcript that represses Ube3a on the paternal chromosome. These results suggest that the repression of human UBE3A-ATS may activate the expression of UBE3A from the paternal chromosome, providing a potential therapeutic strategy for patients with Angelman syndrome.

Structures and molecular mechanisms for common 15q13.3 microduplications involving CHRNA7: benign or pathological?

We have investigated four ∼1.6‐Mb microduplications and 55 smaller 350–680‐kb microduplications at 15q13.2–q13.3 involving the CHRNA7 gene that were detected by clinical microarray analysis. Applying high‐resolution array‐CGH, we mapped all 118 chromosomal breakpoints of these microduplications. We also sequenced 26 small microduplication breakpoints that were clustering at hotspots of nonallelic homologous recombination (NAHR). All four large microduplications likely arose by NAHR between BP4 and BP5 LCRs, and 54 small microduplications arose by NAHR between two CHRNA7‐LCR copies. We identified two classes of ∼1.6‐Mb microduplications and five classes of small microduplications differing in duplication size, and show that they duplicate the entire CHRNA7. We propose that size differences among small microduplications result from preexisting heterogeneity of the common BP4–BP5 inversion. Clinical data and family histories of 11 patients with small microduplications involving CHRNA7 suggest that these microduplications might be associated with developmental delay/mental retardation, muscular hypotonia, and a variety of neuropsychiatric disorders. However, we conclude that these microduplications and their associated potential for increased dosage of the CHRNA7‐encoded α7 subunit of nicotinic acetylcholine receptors are of uncertain clinical significance at present. Nevertheless, if they prove to have a pathological effects, their high frequency could make them a common risk factor for many neurobehavioral disorders. Hum Mutat 31:1–11, 2010.

Iron deficiency in the rat: biochemical studies of brain metabolism.

Summary: Studies were performed to determine the effects of iron deficiency on brain metabolism in rats. Concentrations of cytochrome pigments, oxidative phosphorylation, and catalase and monoamine oxidase activities in brain tissue were unaffected by iron deficiency. However, activities of aldehyde oxidase, a key enzyme in the pathway of serotonin degradation, were significantly reduced, and concentrations of serotonin and total 5-hydroxyindole compounds were elevated in brain tissue of iron-deficient animals. Aldehyde oxidase activities and concentrations of 5-hydroxyindole compounds in brain tissues returned to approximately normal values one week after treatment of iron deficient animals with iron dextran.Speculation: States of iron deficiency may result in reduction of important iron containing enzymes in brain tissue and altered brain metabolism.

Small noncoding differentially methylated copy-number variants, including lncRNA genes, cause a lethal lung developmental disorder

An unanticipated and tremendous amount of the noncoding sequence of the human genome is transcribed. Long noncoding RNAs (lncRNAs) constitute a significant fraction of non-protein-coding transcripts; however, their functions remain enigmatic. We demonstrate that deletions of a small noncoding differentially methylated region at 16q24.1, including lncRNA genes, cause a lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV), with parent-of-origin effects. We identify overlapping deletions 250 kb upstream of FOXF1 in nine patients with ACD/MPV that arose de novo specifically on the maternally inherited chromosome and delete lung-specific lncRNA genes. These deletions define a distant cis-regulatory region that harbors, besides lncRNA genes, also a differentially methylated CpG island, binds GLI2 depending on the methylation status of this CpG island, and physically interacts with and up-regulates the FOXF1 promoter. We suggest that lung-transcribed 16q24.1 lncRNAs may contribute to long-range regulation of FOXF1 by GLI2 and other transcription factors. Perturbation of lncRNA-mediated chromatin interactions may, in general, be responsible for position effect phenomena and potentially cause many disorders of human development.