Recognition of distinct phenotypic features is an important component of genetic diagnosis. Although CHARGE syndrome, Kabuki syndrome, and a recently delineated KMT2D Ex 38/39 allelic disorder exhibit significant overlap, differences on neuroimaging may help distinguish these conditions and guide genetic testing and variant interpretation. We present an infant clinically diagnosed with CHARGE syndrome but subsequently found to have a de novo missense variant in exon 38 of KMT2D, the gene implicated in both Kabuki syndrome and a distinct KMT2D allelic disorder. We compare her brain and inner ear morphology to a retrospective cohort of 21 patients with classic Kabuki syndrome and to typical CHARGE syndrome findings described in the literature. Thirteen of the 21 Kabuki syndrome patients had temporal bone imaging (5/13 CT, 12/13 MRI) and/or brain MRI (12/13) which revealed findings distinct from both CHARGE syndrome and the KMT2D allelic disorder. Our findings further elucidate the spectrum of inner ear dysmorphology distinguishing Kabuki syndrome and the KMT2D allelic disorder from CHARGE syndrome, suggesting that these three disorders may be differentiated at least in part by their inner ear anomalies.

Importance A targeted genomic sequencing platform focused on diseases presenting in the first year of life may minimize financial and ethical challenges associated with rapid whole-genomic sequencing. Objective To report interim variants and associated interpretations of an ongoing study comparing rapid whole-genomic sequencing with a novel targeted genomic platform composed of 1722 actionable genes targeting disorders presenting in infancy. Design, Setting, and Participants The Genomic Medicine in Ill Neonates and Infants (GEMINI) study is a prospective, multicenter clinical trial with projected enrollment of 400 patients. The study is being conducted at 6 US hospitals. Hospitalized infants younger than 1 year of age suspected of having a genetic disorder are eligible. Results of the first 113 patients enrolled are reported here. Patient recruitment began in July 2019, and the interim analysis of enrolled patients occurred from March to June 2020. Interventions Patient (proband) and parents (trios, when available) were tested simultaneously on both genomic platforms. Each laboratory performed its own phenotypically driven interpretation and was blinded to other results. Main Outcomes and Measures Variants were classified according to the American College of Medical Genetics and Genomics standards of pathogenic (P), likely pathogenic (LP), or variants of unknown significance (VUS). Chromosomal and structural variations were reported by rapid whole-genomic sequencing. Results Gestational age of 113 patients ranged from 23 to 40 weeks and postmenstrual age from 27 to 83 weeks. Sixty-seven patients (59%) were male. Diagnostic and/or VUS were returned for 51 patients (45%), while 62 (55%) had negative results. Results were concordant between platforms in 83 patients (73%). Thirty-seven patients (33%) were found to have a P/LP variant by 2 or both platforms and 14 (12%) had a VUS possibly related to phenotype. The median day of life at diagnosis was 22 days (range, 3-313 days). Significant alterations in clinical care occurred in 29 infants (78%) with a P/LP variant. Incidental findings were reported in 7 trios. Of 51 positive cases, 34 (67%) differed in the reported result because of technical limitations of the targeted platform, interpretation of the variant, filtering discrepancies, or multiple causes. Conclusions and Relevance As comprehensive genetic testing becomes more routine, these data highlight the critically important variant detection capabilities of existing genomic sequencing technologies and the significant limitations that must be better understood.

Polycystic kidney disease (PKD) is a condition typified by numerous renal cysts and enlarged kidneys. Types of PKD are generally distinguished by the genetic mode of inheritance, either autosomal dominant (ADPKD) or autosomal recessive (ARPKD). In addition, ADPKD and ARPKD are characterized by differences in clinical and pathological presentations. Whereas ADPKD presents with bilateral renal enlargement and macrocysts, extrarenal cysts (hepatic), intracranial aneurysms, mitral valve prolapse, and a biphasic pattern of progression to end-stage renal disease (ESRD) in later decades of life, ARPKD is characterized by early and rapid enlargement of the kidneys, childhood progression to ESRD, and frequent liver involvement leading to congenital hepatic fibrosis. Because of these differences, family history and clinical presentation are the primary factors used to direct the genetic testing ordered to establish a molecular diagnosis. When a family history includes suspected ADPKD affecting individuals in 1 or more generations, sequencing and/or deletion/duplication analysis of PKD1 and PKD2 is considered. When a family history includes a child with either prenatal or early pediatric onset of PKD, sequencing and/or deletion/duplication analysis of PKHD1 is pursued. Although pathogenic variants in PKD1 and PKD2 account for nearly all PKD cases that appear to be AD, pathogenic variants detected in PKHD1 account for only ∼75% of PKD cases that appear to be AR.1Melchionda S. Palladino T. Castellana S. et al.Expanding the mutation spectrum in 130 probands with ARPKD: identification of 62 novel PKHD1 mutations by Sanger sequencing and MLPA analysis.J Hum Genet. 2016; 61: 811-821Crossref PubMed Scopus (12) Google Scholar Historically, it has been unclear whether these ostensibly AR cases of PKD without detected PKHD1 pathogenic variants represent atypical early manifestations of ADPKD or whether there are yet-unknown genes responsible for an early-onset AR presentation of PKD. Although this landscape is rapidly changing with increasing use of massively parallel sequencing in the form of either gene panels or exome sequencing, particularly in a research setting, clinical practice has been slow to change—although many providers now offer gene panels for evaluation, particularly in the neonatal setting. Research to further understand this phenomenon has yielded important information about the mechanism of disease in PKD. Historically, a "2-hit" hypothesis was supported based on the combination of germline and somatic mutations seen in cysts and the severe, lethal phenotype of homozygous knockout Pkd1 mice.2Reeders S.T. Multilocus polycystic disease.Nat Genet. 1992; 1: 235-237Crossref PubMed Scopus (80) Google Scholar, 3Lu W. Peissel B. Babakhanlou H. et al.Perinatal lethality with kidney and pancreas defects in mice targeted PKD1 mutation.Nat Genet. 1997; 17: 179-181Crossref PubMed Scopus (361) Google Scholar, 4Pei Y. Watnick T. He N. et al.Somatic PKD2 mutations in individual kidney and liver cysts support a 'two-hit' model of cystogenesis in type 2 autosomal dominant polycystic kidney disease.J Am Soc Nephrol. 1999; 10: 1524-1529Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar However, further research in mice with reduced Pkd1 expression has established a dose-dependent model of disease, suggesting that modifier alleles may contribute to the severity and onset of cyst development.5Lantinga-van Leeuwen I.S. Dauwerse J.G. Baelde H.J. et al.Lowering of Pkd1 expression is sufficient to cause polycystic kidney disease.Hum Mol Genet. 2004; 13: 3069-3077Crossref PubMed Scopus (236) Google Scholar,6Hopp K. Ward C.J. Hommerding C.J. et al.Functional polycystin-1 dosage governs autosomal dominant polycystic kidney disease severity.J Clin Invest. 2012; 122: 4257-4273Crossref PubMed Scopus (194) Google Scholar Multiple studies of families with ADPKD have implicated variants in PKD1, PKD2, PKHD1, or HNF1B inherited in trans with known pathogenic PKD1 variants as a cause of earlier-onset, more severe cystic kidney disease.7Rossetti S. Kubly V.J. Consugar M.B. et al.Incompletely penetrant PKD1 alleles suggest a role for gene dosage in cyst initiation in polycystic kidney disease.Kidney Int. 2009; 75: 848-855Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 8Bergmann C. von Bothmer J. Brüchle N.O. et al.Mutations in multiple PKD genes may explain early and severe polycystic kidney disease.J Am Soc Nephrol. 2011; 22: 2047-2056Crossref PubMed Scopus (153) Google Scholar, 9Gilbert R.D. Sukhtankar P. Lachlan K. et al.Bilineal inheritance of PKD1 abnormalities mimicking autosomal recessive polycystic disease.Pediatr Nephrol. 2013; 28: 2217-2220Crossref PubMed Scopus (11) Google Scholar,S1 In addition to identifying hypomorphic alleles that can account for some of the variable expressivity seen in ADPKD families, it has been proposed that the inheritance of 2 such hypomorphic alleles in trans may also cause disease in humans.S2 Here we report a family with 2 siblings diagnosed with PKD in utero, both found to have biallelic inherited mutations in PKD1. The proband was a male fetus found at 20 3/7 weeks gestational age (GA) to have bilateral enlarged, cystic kidneys during an otherwise unremarkable pregnancy with no family history of renal cysts or other features of PKD (Figure 1a). The infant was delivered at 30 6/7 weeks GA due to the development of fetal hydrops in the setting of oligohydramnios and unfortunately died in the delivery room (Figure 1b). Post mortem sequencing and deletion/duplication analysis of PKHD1 was negative. The parents of the proband had a subsequent pregnancy with a male fetus affected with echogenic kidneys at 18 weeks GA, and enlarged, cystic kidneys at 23 1/7 weeks GA. The infant was delivered at 37 weeks GA and was postnatally confirmed to have enlarged echogenic kidneys with innumerable cysts (Figure 1c). He is currently alive at 22 months, and his kidneys remain enlarged with microcysts, but they have not grown rapidly. Exome sequencing was performed on DNA samples from the proband and parents (see Supplementary Methods). Biallelic variants in PKD1 were identified in the proband, and Sanger sequencing using standard techniques identified both variants in the other affected male child (RefSeq: NM_001009944): a maternally inherited missense variant (c.377C>T, p.Pro126Leu) and a paternally inherited missense variant (c.6656C>T, p.Pro2219Leu) (Figure 1d). Both variants are absent from ExAC and gnomAD databases, predicted to be deleterious by in silico models (maternal variant: CADD score 25, paternal variant: CADD score 29, both variants assessed as "probably damaging" by Polyphen, "damaging" by SIFT, "disease causing" by MutationTaster), and the amino acid residues are highly conserved in vertebrates. Neither variant has been previously reported in the literature. Interestingly, PKD1 occurs in a segmentally duplicated portion of the genome. Thus, detecting variants using exome sequencing is complicated by the presence of multiple homologous pseudogenes, which limits the sensitivity of this modality, although the specificity remains highS3; our variants appeared to be of high quality and were Sanger confirmed in the similarly affected sibling. Although both variants are formally classified as variants of uncertain significance, we propose them as disease causing for our family, as we are proposing a novel mechanism and mode of inheritance (as opposed to the typical single loss-of-function variants seen in ADPKD). We further evaluated the exome data for rare, predicted damaging variants in known ARPKD genes such as PKD2, PKHD1, and HNF1B and did not find any candidate variants. We present a case of early-onset PKD attributed to biallelic variants in PKD1. After the proband's variants were identified, we believed this combination of variants to be lethal; however, the live-born second child with the same variants exhibits an attenuated phenotype with an unclear prognosis. At this time, the second affected child is thriving with normal renal function and normal blood pressure despite enlarged cystic kidneys that appear consistent with the phenotype of ARPKD. Although ARPKD is classically associated with biallelic variants in PKHD1, hypomorphic variants in multiple genes in trans with pathogenic variants in PKD1/PKD2 have been found to cause a more severe cystic kidney disease presentation in ADPKD families.7Rossetti S. Kubly V.J. Consugar M.B. et al.Incompletely penetrant PKD1 alleles suggest a role for gene dosage in cyst initiation in polycystic kidney disease.Kidney Int. 2009; 75: 848-855Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar,8Bergmann C. von Bothmer J. Brüchle N.O. et al.Mutations in multiple PKD genes may explain early and severe polycystic kidney disease.J Am Soc Nephrol. 2011; 22: 2047-2056Crossref PubMed Scopus (153) Google Scholar One such hypomorphic variant in PKD1, p.R3277C, has been found recurrently to account for early onset of disease in ADPKD individuals with cyst development in the pediatric period.7Rossetti S. Kubly V.J. Consugar M.B. et al.Incompletely penetrant PKD1 alleles suggest a role for gene dosage in cyst initiation in polycystic kidney disease.Kidney Int. 2009; 75: 848-855Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar,S2 Although this recurrent hypomorphic variant has not been reported in a human in a homozygous state, it has been proposed that the inheritance of 2 such hypomorphic alleles in trans may also cause disease in humans. In a couple with 2 children diagnosed with PKD in utero with no PKHD1 variants identified, analysis of PKD1 and PKD2 revealed that each healthy parent carried a known or suspected hypomorphic PKD1 variant, and each affected child had inherited these mutations in trans.S2 Our case is the second family documented with PKD in utero caused by biallelic PKD1 hypomorphic variants, although phenotypic variability is seen within this family, as described above. Given the current evidence, we propose that compound heterozygous or homozygous hypomorphic variants in PKD1 may represent a proportion of early-onset PKD cases that are not accounted for by either biallelic variants in PKHD1 or null mutations in PKD1/PKD2 modified by a hypomorphic allele in another PKD-related gene. Because of the demonstrated variability in genes that can contribute to early-onset PKD, broadening the genetic testing approach taken toward a molecular diagnosis could benefit families affected by early-onset PKD. In a recent study of 36 individuals with a clinical diagnosis of ARPKD, 8 (22%) did not carry PKHD1 variants and instead carried variants in other kidney disease−associated genes.S4 Although recommendations have been made against single-gene analysis of PKHD1 as a first-line diagnostic approach in light of the contribution of additional genes to phenocopy disorders,S5 this was a standard practice for many years and continues to be practiced in some clinical settings. In order to move toward more comprehensive genetic testing for early-onset PKD, the historical model delineating early- and later-onset PKD by gene(s) needs to be reconsidered. The current model of defining ADPKD by 1 pathogenic variant in PKD1 or PKD2 and ARPKD by 2 pathogenic variants in PKHD1 no longer encompasses what is known about the dose-dependent mechanism responsible for disease development and the multitude of genes involved. A model of describing early-onset PKD as 2 variants in PKD-related genes (PKD1, PKD2, PKHD1, HNF1B) and later-onset PKD as 1 variant in a PKD-related gene would more accurately represent our knowledge of the underlying causes of PKD. TEM is an employee of Quest Diagnostics. All the other authors declared no competing interests. MHW received support from T32 GM007748. Sequencing and analysis were provided by the Broad Institute of MIT and Harvard Center for Mendelian Genomics (Broad CMG) and was funded by the National Human Genome Research Institute , the National Eye Institute , and the National Heart, Lung, and Blood Institute grant UM1HG008900 and in part by National Human Genome Research Institute grant R01 HG009141 . The authors wish to thank the family for their participation in the research. Download .pdf (.11 MB) Help with pdf files Supplementary File (PDF)

Purpose Several hundred genetic muscle diseases have been described, all of which are rare. Their clinical and genetic heterogeneity means that a genetic diagnosis is challenging. We established an international consortium, MYO-SEQ, to aid the work-ups of muscle disease patients and to better understand disease etiology. Methods Exome sequencing was applied to 1001 undiagnosed patients recruited from more than 40 neuromuscular disease referral centers; standardized phenotypic information was collected for each patient. Exomes were examined for variants in 429 genes associated with muscle conditions. Results We identified suspected pathogenic variants in 52% of patients across 87 genes. We detected 401 novel variants, 116 of which were recurrent. Variants in CAPN3 , DYSF , ANO5 , DMD , RYR1 , TTN , COL6A2 , and SGCA collectively accounted for over half of the solved cases; while variants in newer disease genes, such as BVES and POGLUT1 , were also found. The remaining well-characterized unsolved patients (48%) need further investigation. Conclusion Using our unique infrastructure, we developed a pathway to expedite muscle disease diagnoses. Our data suggest that exome sequencing should be used for pathogenic variant detection in patients with suspected genetic muscle diseases, focusing first on the most common disease genes described here, and subsequently in rarer and newly characterized disease genes.

OBJECTIVE:To determine the proportion of infant deaths occurring in the setting of a confirmed genetic disorder.STUDY DESIGN:A retrospective analysis of the electronic medical records of infants born from 1 January, 2011 to 1 June, 2017, who died prior to 1 year of age.RESULTS:Five hundred and seventy three deceased infants were identified. One hundred and seventeen were confirmed to have a molecular or cytogenetic diagnosis in a clinical diagnostic laboratory and an additional seven were diagnosed by research testing for a total of 124/573 (22%) diagnosed infants. A total of 67/124 (54%) had chromosomal disorders and 58/124 (47%) had single gene disorders (one infant had both). The proportion of diagnoses made by sequencing technologies, such as exome sequencing, increased over the years.CONCLUSIONS:The prevalence of confirmed genetic disorders within our cohort of infant deaths is higher than that previously reported. Increased efforts are needed to further understand the mortality burden of genetic disorders in infancy.

Background The eMERGE III Network was tasked with harmonizing genetic testing protocols linking multiple sites and investigators.Methods DNA capture panels targeting 109 genes and 1551 variants were constructed by two clinical sequencing centers for analysis of 25,000 participant DNA samples collected at 11 sites where samples were linked to patients with electronic health records. Each step from sample collection, data generation, interpretation, reporting, delivery and storage, were developed and validated in CAP/CLIA settings and harmonized across sequencing centers.Results A compliant and secure network was built and enabled ongoing review and reconciliation of clinical interpretations while maintaining communication and data sharing between investigators. Mechanisms for sustained propagation and growth of the network were established. An interim data freeze representing 15,574 sequenced subjects, informed the assay performance for a range of variant types, the rate of return of results for different phenotypes and the frequency of secondary findings. Practical obstacles for implementation and scaling of clinical and research findings were identified and addressed. The eMERGE protocols and tools established are now available for widespread dissemination.Conclusions This study established processes for different sequencing sites to harmonize the technical and interpretive aspects of sequencing tests, a critical achievement towards global standardization of genomic testing. The network established experience in the return of results and the rate of secondary findings across diverse biobank populations. Furthermore, the eMERGE network has accomplished integration of structured genomic results into multiple electronic health record systems, setting the stage for clinical decision support to enable genomic medicine.

The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.

Dystroglycanopathies are a clinically and genetically heterogeneous group of disorders that are typically characterised by limb-girdle muscle weakness. Mutations in 18 different genes have been associated with dystroglycanopathies, the encoded proteins of which typically modulate the binding of α-dystroglycan to extracellular matrix ligands by altering its glycosylation. This results in a disruption of the structural integrity of the myocyte, ultimately leading to muscle degeneration.

Ribosomal RNA (rRNA) is transcribed from rDNA by RNA polymerase I (Pol I) to produce the 45S precursor of the 28S, 5.8S, and 18S rRNA components of the ribosome. Two transcription factors have been defined for Pol I in mammals, the selectivity factor SL1, and the upstream binding transcription factor (UBF), which interacts with the upstream control element to facilitate the assembly of the transcription initiation complex including SL1 and Pol I. In seven unrelated affected individuals, all suffering from developmental regression starting at 2.5-7 years, we identified a heterozygous variant, c.628G>A in UBTF, encoding p.Glu210Lys in UBF, which occurred de novo in all cases. While the levels of UBF, Ser388 phosphorylated UBF, and other Pol I-related components (POLR1E, TAF1A, and TAF1C) remained unchanged in cells of an affected individual, the variant conferred gain of function to UBF, manifesting by markedly increased UBF binding to the rDNA promoter and to the 50-external transcribed spacer. This was associated with significantly increased 18S expression, and enlarged nucleoli which were reduced in number per cell. The data link neurodegeneration in childhood with altered rDNA chromatin status and rRNA metabolism.

Gain-of-function variants in some RAS–MAPK pathway genes, including PTPN11 and NRAS, are associated with RASopathies and/or acquired hematological malignancies, most notably juvenile myelomonocytic leukemia (JMML). With rare exceptions, the spectrum of germline variants causing RASopathies does not overlap with the somatic variants identified in isolated JMML. Studies comparing these variants suggest a stronger gain-of-function activity in the JMML variants. As JMML variants have not been identified as germline defects and have a greater impact on protein function, it has been speculated that they would be embryonic lethal. Here we identified three variants, which have previously only been identified in isolated somatic JMML and other sporadic cancers, in four cases with a severe pre- or neo-natal lethal presentation of Noonan syndrome. These cases support the hypothesis that these stronger gain-of-function variants are rarely compatible with life.

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD, little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD, we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially, no significant phenotypic differences were observed between iPSCs derived from the different family members. However, upon directed neural differentiation, we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared with their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity, including WNT pathway components and ion channel subunits. Treatment of the CXCR4(+) NPCs with a pharmacological inhibitor of glycogen synthase kinase 3, a known regulator of WNT signaling, was found to rescue a progenitor proliferation deficit in the BD patient NPCs. Taken together, these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention.

mRNA synthesis, processing, and destruction involve a complex series of molecular steps that are incompletely understood. Because the RNA intermediates in each of these steps have finite lifetimes, extensive mechanistic and dynamical information is encoded in total cellular RNA. Here we report the development of SnapShot-Seq, a set of computational methods that allow the determination of in vivo rates of pre-mRNA synthesis, splicing, intron degradation, and mRNA decay from a single RNA-Seq snapshot of total cellular RNA. SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA. We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors. SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment.

Norrie disease (ND) is an X‐linked recessive disorder characterized by congenital blindness, progressive sensorineural hearing loss and cognitive impairment. The ocular phenotype has been well described, while the extraocular manifestations of the disorder are not well understood. We present the data from the Norrie Disease Registry, which consists of 56 patients with detailed clinical histories and genotype data. This study represents the largest, detailed investigation into the phenotypic spectrum of ND to date and more importantly expands knowledge of the extraocular clinical manifestations. We identify several novel aspects of the syndrome that will improve the management of these patients. In particular, we expand our understanding of the neurologic manifestations in ND and identify a chronic seizure disorder in approximately 10% of all patients. In addition, details of the hearing phenotype are described including the median age of onset (12 years of age) and how genotype affects onset. Moreover, we find vascular disease to be a significant component of ND; and vascular health should be, in the future, a component of patient clinical care. In summary, the results expand our understanding of the phenotypic variability and genotypic heterogeneity in ND patients. © 2012 Wiley Periodicals, Inc.

SOD1, ANG, TARDBP and FUS mutations have been associated with amyotrophic lateral sclerosis (ALS). Our goal was to extend molecular genetic analysis to newly identified ALS genetic loci and to determine the frequency of mutations, distribution of disease genes, and variant spectrum of these genes in a large United States ALS-phenotype cohort. We screened 1220 probands with an ALS phenotype, referred originally for SOD1 molecular genetic analysis. 1128 SOD1-negative probands were screened for ANG, and 277 and 223 SOD1- and ANG-negative samples were screened for TARDBP and FUS, respectively. One hundred additional probands were specifically screened only for FUS exon 15. We identified a total of 36 different SOD1 mutations, including three novel mutations, in 92 probands. ANG screening identified three mutations, including two novel mutations, and TARDBP screening identified two previously reported TARDBP mutations. We also identified four mutations in FUS, including the reported FUS in-frame deletion, c.430_447del, p.Gly144_Tyr149del, in a patient with inclusion body myositis, and two known FUS missense mutations. From this study, we estimate frequencies for SOD1, ANG, TARDBP and FUS mutations, in this United States cohort, to be 7.5%, 0.71%, 0.72% and 1.9%, respectively. In conclusion, we identify novel variants in SOD1, ANG, TARDBP and FUS, and expand the FUS-associated clinicopathologic phenotype.

OBJECTIVES:To explore a potential expansion of the phenotypic and genotypic characteristics of Finnish variant late-infantile neuronal ceroid lipofuscinosis (NCL), we screened a collection of 47 patients with clinically diagnosed NCL in whom no molecular diagnosis had been made.METHODS:We used PCR amplification of genomic DNA, followed by fluorescent-labeled dideoxy-nucleotide chain termination sequencing and multiplex ligation-dependent probe amplification, to screen our cohort of patients for mutations in CLN5. We collected ethnic background, clinical, and pathologic information, as available, to clarify the breadth of CLN5 disease expression and to explore possible genotype-phenotype correlations.RESULTS:We identified 10 patients with pathogenic CLN5 mutations, including 11 mutations not previously described: 4 missense, 5 out-of-frame insertion/deletion mutations, and 2 large intragenic deletions. We also documented 3 previously reported CLN5 mutations. The age at disease onset in this cohort is predominantly juvenile rather than late infantile. Importantly, we have identified 2 adult-onset patients who share a common pathogenic allele. The majority of patients presented with motor and visual impairments and not seizures. In those patients with available longitudinal data, most had progressed to global neurodevelopmental and visual failure with seizures within 1 to 4 years.CONCLUSIONS:Our study suggests that CLN5 mutations 1) are more common in patients with neuronal ceroid lipofuscinosis (NCL) than previously reported, 2) are found in non-Finnish NCL patients of broad ethnic diversity, and 3) can be identified in NCL patients with disease onset in adult and juvenile epochs. CLN5 genetic testing is warranted in a wider population with clinical and pathologic features suggestive of an NCL disorder.

Histone mRNAs are the only eukaryotic cellular mRNAs that are not polyadenylated. Synthesis of mature histone mRNA requires only a single processing reaction: an endonucleolytic cleavage between a conserved stem-loop and a purine-rich downstream element to form the 3' end. The stem-loop binding protein (SLBP) is required for processing, and following processing, histone mRNA is transported to the cytoplasm, where SLBP participates in translation of the histone mRNA and is also involved in regulation of histone mRNA degradation. Here we present an analysis of histone mRNA metabolism in cells with highly reduced levels of SLBP using RNA interference. Knocking down SLBP in U2OS cells results in a reduction in the rate of cell growth and an accumulation of cells in S-phase. Surprisingly, there is only a modest (twofold) decrease in histone mRNA levels. Much of histone mRNA in the SLBP knockdown cells is properly processed but is retained in the nucleus. The processed histone mRNA in SLBP knockdown cells is not rapidly degraded when DNA replication is inhibited. These results suggest a previously undescribed role for SLBP in histone mRNA export.

Histone mRNAs are rapidly degraded at the end of S phase or when DNA replication is inhibited. Histone mRNAs end in a conserved stem-loop rather than a poly(A) tail. Degradation of histone mRNAs requires the stem-loop sequence, which binds the stem-loop-binding protein (SLBP), active translation of the histone mRNA, and the location of the stem-loop close to the termination codon. We report that the initial step in histone mRNA degradation is the addition of uridines to the 3' end of the histone mRNA, both after inhibition of DNA replication and at the end of S phase. Lsm1 is required for histone mRNA degradation and is present in a complex containing SLBP on the 3' end of histone mRNA after inhibition of DNA replication. We cloned degradation intermediates that had been partially degraded from both the 5' and the 3' ends. RNAi experiments demonstrate that both the exosome and 5'-to-3' decay pathway components are required for degradation, and individual histone mRNAs are then degraded simultaneously 5' to 3' and 3' to 5'.

The replication‐dependent histone mRNAs are the only eukaryotic mRNAs that are not polyadenylated, ending instead in a conserved stem‐loop. Histone mRNAs are present in high levels only in S‐phase, and they are regulated coordinately with DNA replication. A major regulatory step is the rapid degradation of histone mRNA when DNA replication is inhibited or at the end of S‐phase. We have elucidated the pathway of histone mRNA degradation. ATR, a kinase activated when DNA synthesis is inhibited, is required for histone mRNA degradation, although the intermediate steps in signaling that ultimately initiate histone mRNA degradation are not known. The stemloop must be close (25–75 nts) to the termination codon for efficient regulation of translation and the mRNA must be actively translated. The protein that binds the stem‐loop, SLBP, is involved in translation and is likely a target of the signaling pathway that activates histone mRNA degradation, as a result of inefficient translation termination. The inefficient termination leads to the recruitment of Upf1, the critical factor also required for nonsense mediated decay. A terminal uridyl transferase (TUTase) is then recruited and 8–12 U's added to the end of the histone mRNA. This provides a platform for binding Lsm1‐7, which in turn recruit the decapping enzymes, as well as the exosome to degrade individual histone molecules simultaneously from both ends.

Replication-dependent histone mRNAs are coordinately regulated in parallel with DNA replication. Histone mRNAs accumulate to high levels only in S-phase cells and are degraded rapidly at the end of S phase or when DNA replication is inhibited in S-phase cells. The unique 3' end on histone mRNAs is the cis element responsible for the regulation of histone mRNA degradation. This chapter describes the approaches used to demonstrate the connection between translation of histone mRNA and its degradation as well as the pathway of histone mRNA degradation in mammalian cells. In particular, the initial step in histone mRNA degradation is attachment of an oligo(U) tail to the 3' end of histone mRNA, providing a platform for binding factors that trigger mRNA degradation.

The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of > 400 cell cycle- regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with the cell cycle phase redistribution after genotoxin treatment, the direct transcriptional target genes were distinguished from genes for which expression changed secondary to cell synchronization. Application of this model to ionizing radiation ( IR)- treated normal human fibroblasts identified 150 of 406 cycle- regulated genes as putative direct transcriptional targets of IR-induced DNA damage. Changes in expression of these genes after IR treatment derived from both direct transcriptional regulation and cell cycle synchronization.