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Disrupting variants in the DMD gene are associated with Duchenne or Becker muscular dystrophy (DMD/BMD) or with hyperCKemia, all of which present very different degrees of clinical severity. The clinical phenotypes of these disorders could not be distinguished in infancy or early childhood. Accurate phenotype prediction based on DNA variants may therefore be required in addition to invasive tests, such as muscle biopsy. Transposon insertion is one of the rarest mutation types. Depending on their position and characteristics, transposon insertions may affect the quality and/or quantity of dystrophin mRNA, leading to unpredictable alterations in gene products. Here, we report the case of a three-year-old boy showing initial skeletal muscle involvement in whom we characterized a transposon insertion (Alu sequence) in exon 15 of the DMD gene. In similar cases, the generation of a null allele is predicted, resulting in a DMD phenotype. However, mRNA analysis of muscle biopsy tissue revealed skipping of exon 15, which restored the reading frame, thus predicting a milder phenotype. This case is similar to very few others already described in the literature. This case further enriches our knowledge of the mechanisms perturbing splicing and causing exon skipping in DMD , helping to properly guide clinical diagnosis.

Mutations in COL4A3-A5 cause a spectrum of glomerular disorders, including thin basement membrane nephropathy (TBMN) and Alport syndrome (AS). The wide application of next-generation sequencing (NGS) in the last few years has revealed that mutations in these genes are not limited to these clinical entities. In this study, 176 individuals with a clinical diagnosis of inherited kidney disorders underwent an NGS-based analysis to address the underlying cause; those who changed or perfected the clinical diagnosis after molecular analysis were selected. In 5 out of 83 individuals reaching a molecular diagnosis, the genetic result was unexpected: three individuals showed mutations in collagen type IV genes. These patients showed the following clinical pictures: (1) familial focal segmental glomerulosclerosis; (2) end-stage renal disease (ESRD) diagnosed incidentally in a 49-year-old man, with diffuse cortical calcifications on renal imaging; and (3) dysmorphic and asymmetric kidneys with multiple cysts and signs of tubule-interstitial defects. Genetic analysis revealed rare heterozygote/compound heterozygote COL4A4-A5 variants. Our study highlights the key role of NGS in the diagnosis of inherited renal disorders and shows the phenotype variability in patients carrying mutations in collagen type IV genes.

Chronic kidney disease (CKD) is a major clinical sign of patients with Bardet-Biedl syndrome (BBS), especially in those carrying BBS10 mutations. Twenty-nine patients with BBS and 30 controls underwent a serum-targeted metabolomic analysis. In vitro studies were conducted in two kidney-derived epithelial cell lines, where Bbs10 was stably deleted (IMCD3- Bbs10 -/-cells) and over-expressed. The CKD status affected plasmatic metabolite fingerprinting in both patients with BBS and controls. Specific phosphatidylcholine and acylcarnitines discriminated eGFR decline only in patients with BBS. IMCD3- Bbs10 -/ cells displayed intracellular lipidaccumulation, reduced mitochondrial potential membrane and citrate synthase staining. Mass-Spectrometry-based analysis revealed that human BBS10 interacted with six mitochondrial proteins, in vitro. In conclusion, renal dysfunction correlated with abnormal phosphatidylcholine and acylcarnitines plasma levels in patients with BBS; in vitro , Bbs10 depletion caused mitochondrial defects while human BBS10 interacted with several mitochondria-related proteins, suggesting an unexplored role of this protein., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Next generation sequencing (NGS) has changed our approach to diagnosis of genetic disorders. Nowadays, the most comprehensive application of NGS is whole genome sequencing (WGS) that is able to detect virtually all DNA variations. However, even after accurate WGS, many genetic conditions remain unsolved. This may be due to the current NGS protocols, based on DNA fragmentation and short reads. To overcome these limitations, we applied a linked-read sequencing technology that combines single-molecule barcoding with short-read WGS. We were able to assemble haplotypes and distinguish between alleles along the genome. As an exemplary case, we studied the case of a female carrier of X-linked muscular dystrophy with an unsolved genetic status. A deletion of exons 16-29 in DMD gene was responsible for the disease in her family, but she showed a normal dosage of these exons by Multiplex Ligation-dependent Probe Amplification (MLPA) and array CGH. This situation is usually considered compatible with a "non-carrier" status. Unexpectedly, the girl also showed an increased dosage of flanking exons 1-15 and 30-34. Using linked-read WGS, we were able to distinguish between the two X chromosomes. In the first allele, we found the 16-29 deletion, while the second allele showed a 1-34 duplication: in both cases, linked-read WGS correctly mapped the borders at single-nucleotide resolution. This duplication in trans apparently restored the normal dosage of exons 16-29 seen by quantitative assays. This had a dramatic impact in genetic counselling, by converting a non-carrier into a double carrier status prediction. We conclude that linked-read WGS should be considered as a valuable option to improve our understanding of unsolved genetic conditions.

Background: Mutations in RAB39B at Xq28 causes a rare form of X-linked intellectual disability (ID) and Parkinson's disease. Neurofibromatosis type 1 (NF1) is caused by heterozygous mutations in NF1 occurring de novo in about 50% of cases, usually due to paternal gonadal mutations. This case report describes clinical and genetic findings in a boy with the occurrence of two distinct causative mutations in NF1 and RAB39B explaining the observed phenotype., Case Presentation: Here we report a 7-year-old boy with multiple café-au-lait macules (CALMs) and freckling, severe macrocephaly, peculiar facial gestalt, severe ID with absent speech, epilepsy, autistic traits, self-harming, and aggressiveness. Proband is an only child born to a father aged 47. Parents did not present signs of NF1, while a maternal uncle showed severe ID, epilepsy, and tremors.By RNA analysis of NF1, we identified a de novo splicing variant (NM_000267.3:c.6579+2T>C) in proband, which explained NF1 clinical features but not the severe ID, behavioral problems, and aggressiveness. Family history suggested an X-linked condition and massively parallel sequencing of X-exome identified a novel RAB39B mutation (NM_171998.2:c.436_447del) in proband, his mother, and affected maternal uncle, subsequently validated by Sanger sequencing in these and other family members., Conclusions: The case presented here highlights how concurrent genetic defects should be considered in NF1 patients when NF1 mutations cannot reasonably explain all the observed clinical features.

A nonsense mutation adds a premature stop signal that hinders any further translation of a protein-coding gene, usually resulting in a null allele. To investigate the possible exceptions, we used the DMD gene as an ideal model. First, because dystrophin absence causes Duchenne muscular dystrophy (DMD), while its reduction causes Becker muscular dystrophy (BMD). Second, the DMD gene is X-linked and there is no second allele that can interfere in males. Third, databases are accumulating reports on many mutations and phenotypic data. Finally, because DMD mutations may have important therapeutic implications. For our study, we analyzed large databases (LOVD, HGMD and ClinVar) and literature and revised critically all data, together with data from our internal patients. We totally collected 2593 patients. Positioning these mutations along the dystrophin transcript, we observed a nonrandom distribution of BMD-associated mutations within selected exons and concluded that the position can be predictive of the phenotype. Nonsense mutations always cause DMD when occurring at any point in fifty-one exons. In the remaining exons, we found milder BMD cases due to early 5' nonsense mutations, if reinitiation can occur, or due to late 3' nonsense when the shortened product retains functionality. In the central part of the gene, all mutations in some in-frame exons, such as in exons 25, 31, 37 and 38 cause BMD, while mutations in exons 30, 32, 34 and 36 cause DMD. This may have important implication in predicting the natural history and the efficacy of therapeutic use of drug-stimulated translational readthrough of premature termination codons, also considering the action of internal natural rescuers. More in general, our survey confirm that a nonsense mutation should be not necessarily classified as a null allele and this should be considered in genetic counselling., Competing Interests: The authors have declared that no competing interests exist.

Pigmentary manifestations can represent an early clinical sign in children affected by Neurofibromatosis type 1 (NF1), Legius syndrome, and other neurocutaneous disorders. The differential molecular diagnosis of these pathologies is a challenge that can now be met by combining next generation sequencing of target genes with concurrent second-level tests, such as multiplex ligation-dependent probe amplification and RNA analysis. We clinically and genetically investigated 281 patients, almost all pediatric cases, presenting with either NF1 ( n = 150), only pigmentary features (café au lait macules with or without freckling; ( n = 95), or clinical suspicion of other RASopathies or neurocutaneous disorders ( n = 36). The causative variant was identified in 239 out of the 281 patients analyzed (85.1%), while 42 patients remained undiagnosed (14.9%). The NF1 and SPRED1 genes were mutated in 73.3% and 2.8% of cases, respectively. The remaining 8.9% carried mutations in different genes associated with other disorders. We achieved a molecular diagnosis in 69.5% of cases with only pigmentary manifestations, allowing a more appropriate clinical management of these patients. Our findings, together with the increasing availability and sharing of clinical and genetic data, will help to identify further novel genotype-phenotype associations that may have a positive impact on patient follow-up.

UBE2A deficiency is a syndromic condition of X-linked intellectual disability (ID) characterized by typical dysmorphic features that include synophrys, prominent supraorbital ridges, almond-shaped, and deep-set eyes, large ears, wide mouth, myxedematous appearance, hirsutism, micropenis, and onychodystrophy. To date, only seven familial UBE2A intragenic mutations and nine larger microdeletions encompassing UBE2A have been reported. Here, we describe two siblings with X-linked ID and typical clinical features of UBE2A deficiency caused by a novel hemizygous variant, identified by massively parallel sequencing of X-exome. The synonymous c.330G>A substitution in UBE2A modifies the last nucleotide of exon 5, causing the exon skipping and resulting in an out-of-frame transcript, likely encoding for a truncated form of the ubiquitin-conjugating enzyme E2 A. As confirmed by deep sequencing, the c.330G>A substitution in UBE2A was undetectable in genomic DNA from maternal blood cells, suggesting that the recurrent UBE2A deficiency observed in males of this family is caused by a maternal germline mosaicism., (© 2017 Wiley Periodicals, Inc.)

Objective: To apply next-generation sequencing (NGS) for the investigation of the genetic basis of undiagnosed muscular dystrophies and myopathies in a very large cohort of patients., Methods: We applied an NGS-based platform named MotorPlex to our diagnostic workflow to test muscle disease genes with a high sensitivity and specificity for small DNA variants. We analyzed 504 undiagnosed patients mostly referred as being affected by limb-girdle muscular dystrophy or congenital myopathy., Results: MotorPlex provided a complete molecular diagnosis in 218 cases (43.3%). A further 160 patients (31.7%) showed as yet unproven candidate variants. Pathogenic variants were found in 47 of 93 genes, and in more than 30% of cases, the phenotype was nonconventional, broadening the spectrum of disease presentation in at least 10 genes., Conclusions: Our large DNA study of patients with undiagnosed myopathy is an example of the ongoing revolution in molecular diagnostics, highlighting the advantages in using NGS as a first-tier approach for heterogeneous genetic conditions., (© 2016 American Academy of Neurology.)

Objective and Importance: To verify the presence of β-thalassemia in subjects showing hematologic phenotype of α-thalassemia, conduct normal molecular sequence analysis of the α-globin genes, and detect the absence of the most frequent α-thalassemia deletions., Clinical Presentation: A patient from Apulia (Southern Italy) was referred to our institution for the occasional founding of hypochromic polyglobulia and microcytic red blood cells associated with normal levels of Hb A2 and Hb F and normal iron parameters., Intervention and Technique: The patient has been investigated using Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), quantitative real-time PCR, restriction analysis, and gap-PCR. A novel deletion, the Italian (ϵγδβ)(0)-thalassemia, has been identified. The 5' breakpoint was within a LINE element of 80 kb 3' of the ε-globin gene, and the 3' breakpoint was within a 160-bp palindrome of about 30 kb 5' of the β-globin gene. The breakpoint region was characterized by the presence of a microhomology (5'-TCT-3') and of an insertion of 43 bp owing to the duplication of the 160-bp palindrome. Comparison of the Hb and Hb A2 values of (ϵγδβ)(0)-thalassemia from the literature with those of (molecularly known) thalassemia carriers indicated a higher level of Hb A2 with respect to α-thalassemia and a lower level of Hb with respect to β(0)-thalassemia carriers., Conclusion: In this study, we report the first (ϵγδβ)(0)-thalassemia case identified in Italy. To avoid misdiagnosis of β-thalassemia, we suggest verifying the presence of large deletions of the β-globin gene cluster in subjects showing a higher border line level of Hb A2 and a lower level of Hb.

Aims: We aimed at evaluating a standard multiplex ligation-dependent probe amplification (MLPA) probe set for the detection of aneuploidy to diagnose Turner syndrome (TS). We first fixed an MLPA ratio cutoff able to detect all cases of TS in a pilot TS group. We then tested this value on a second group of TS patients and a short-stature population to measure specificity and sensitivity., Methods: 15 TS patients with X mosaicism or X structural abnormalities (Pilot TS Group), 45 TS karyotype-assessed patients (TS Group), and 74 prepubertal female patients with apparent idiopathic short stature (Short-Stature Group) were enrolled. All subjects underwent MLPA and karyotype analysis. In the TS and Short-Stature Groups, MLPA testing was performed in blind., Results: The choice of an MLPA threshold ratio of 0.76 for at least 1 probe allowed us to detect all TS cases, including mosaicisms. Sensitivity and specificity were 100% (CI 95%, 0.92-1) and 88.89% (CI 95%, 0.79-0.94), respectively. The positive predictive value was 88.5%, and the negative predictive value was 100%. MLPA detected the presence of Y chromosome material in 2 patients., Conclusion: MLPA is an accurate and inexpensive tool to screen for TS in girls with short stature. A customized MLPA kit may be useful for the screening of an even larger population., (© 2016 S. Karger AG, Basel.)

Limb-girdle muscular dystrophies (LGMD) are genetically and clinically heterogeneous conditions. We investigated a large family with autosomal dominant transmission pattern, previously classified as LGMD1F and mapped to chromosome 7q32. Affected members are characterized by muscle weakness affecting earlier the pelvic girdle and the ileopsoas muscles. We sequenced the whole exome of four family members and identified a shared heterozygous frame-shift variant in the Transportin 3 (TNPO3) gene, encoding a member of the importin-β super-family. The TNPO3 gene is mapped within the LGMD1F critical interval and its 923-amino acid human gene product is also expressed in skeletal muscle. In addition, we identified an isolated case of LGMD with a new missense mutation in the same gene. We localized the mutant TNPO3 around the nucleus, but not inside. The involvement of gene related to the nuclear transport suggests a novel disease mechanism leading to muscular dystrophy.

Background: Array-based comparative genomic hybridization (aCGH) is a reference high-throughput technology for detecting large pathogenic or polymorphic copy-number variations in the human genome; however, a number of quantitative monogenic mutations, such as smaller heterozygous deletions or duplications, are usually missed in most disease genes when proper multiplex ligation-dependent probe assays are not performed., Methods: We developed the Motor Chip, a customized CGH array with exonic coverage of 245 genes involved in neuromuscular disorders (NMDs), as well as 180 candidate disease genes. We analyzed DNA samples from 26 patients with known deletions or duplications in NMDs, 11 patients with partial molecular diagnoses, and 19 patients with a clinical diagnosis alone., Results: The Motor Chip efficiently confirmed and refined the copy-number mutations in all of the characterized patients, even when only a single exon was involved. In noncharacterized or partially characterized patients, we found deletions in the SETX (senataxin), SGCG [sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein)], and LAMA2 (laminin, alpha 2) genes, as well as duplications involving LAMA2 and the DYSF [dysferlin, limb girdle muscular dystrophy 2B (autosomal recessive)] locus., Conclusions: The combination of exon-specific gene coverage and optimized platform and probe selection makes the Motor Chip a complementary tool for molecular diagnosis and gene investigation in neuromuscular diseases.

A second genetic revolution is approaching thanks to next-generation DNA sequencing technologies. In the next few years, the 1,000$-genome sequencing promises to reveal every individual variation of DNA. There is, however, a major problem: the identification of thousands of nucleotide changes per individual with uncertain pathological meaning. This is also an ethical issue. In the middle, there is today the possibility to address the sequencing analysis of genetically heterogeneous disorders to selected groups of genes with defined mutation types. This will be cost-effective and safer. We assembled an easy-to manage overview of most Mendelian genes involved in myopathies, cardiomyopathies, and neuromyopathies. This was entirely put together using a number of open access web resources that are listed below. During this effort we realized that there are unexpected countless sources of data, but the confusion is huge. In some cases, we got lost in the validation of disease genes and in the difficulty to discriminate between polymorphisms and disease-causing alleles. In the table are the annotated genes, their associated disorders, genomic, mRNA and coding sizes. We also counted the number of pathological alleles so far reported and the percentage of single nucleotide mutations.

PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.

The amino acid sequence of a novel tissue-and phase-specific nuclear protein (SNP) has been determined, after purification from the nuclei of the oviduct of the lizard Podarcis sicula Raf. during the reproductive period of the seasonal growth. SNP has a pI of 9.0 and contains 81 amino acid residues with a molecular weight of 9211.88 +/- 0.09. It shows a bipartite organization as the first 40 amino acids contain all 8 cysteinyl residues, while the last 41 amino acids contain 16 prolyl residues. Two more components have also been identified and characterized, with the first 79 amino acids matching SNP and missing one or two residues at the C-terminus. They have thus been named [des-(Ala81) SNP1] and [des-(Lys80-Ala81) SNP2], respectively. The molecular weights are 9140.21 +/- 0.83 for [des-(Ala81) SNP1] and 9011 +/- 0.09 for [des-(Lys80-Ala81) SNP2].

Phytolacca dioica L. leaves produce at least two type-I ribosome-inactivating proteins. Each polypeptide chain is subjected to different post-translational modifications giving rise to PD-L1 and PD-L2, and PD-L3 and PD-L4, each polypeptide pair having the same primary structure. With the aim of exploiting the cytotoxic properties of these proteins as potential biological phytodrugs, a gene encoding PD-L4 was designed based on criteria expected to maximize the translation efficiency in tomato. The gene was constructed from 18 oligonucleotides and preliminarily expressed in Escherichia coli, using the T7 promoter system. The protein produced was insoluble and accumulated in inclusion bodies to about 300 mg/l of culture. Ribosome-inactivating activity was generated by controlled oxidation of the reduced and denatured protein. The recombinant protein was indistinguishable from natural PD-L4 as isolated from leaves of Phytolacca dioica, in both catalytic activity and primary structure.

New single-chain (type 1) ribosome-inactivating proteins (RIPs) were isolated from the seeds of Basella rubra L. (two proteins) and from the leaves of Bougainvillea spectabilis Willd. (one protein). These RIPs inhibit protein synthesis both in a cell-free system, with an IC50 (concentration causing 50% inhibition) in the 10(-10) M range, and by various cell lines, with IC50S in the 10(-8)-10(-6) M range. All three RIPs released adenine not only from rat liver ribosomes but also from Escherichia coli rRNA, polyadenylic acid, herring sperm DNA, and artichoke mottled crinkle virus (AMCV) genomic RNA, thus being polynucleotide:adenosine glycosidases. The proteins from Basella rubra had toxicity to mice similar to that of most type 1 RIPs (Barbieri et al., 1993, Biochim Biophys Acta 1154: 237-282) with an LD50 (concentration that is 50% lethal) < or = 8 mg.kg-1 body weight, whilst the RIP from Bougainvillea spectabilis had an LD50 > 32 mg.kg-1. The N-terminal sequence of the two RIPs from Basella rubra had 80-93% identity, whereas it differed from the sequence of the RIP from Bougainvillea spectabilis. When tested with antibodies against various RIPs, the RIPs from Basella gave some cross-reactivity with sera against dianthin 32, and weak cross-reactivity with momordin I and momorcochin-S, whilst the RIP from Bougainvillea did not cross-react with any antiserum tested. An RIP from Basella rubra and one from Bougainvillea spectabilis were tested for antiviral activity, and both inhibited infection of Nicotiana benthamiana by AMCV.

The primary structure has been determined for PD-S2, a new type 1 ribosome-inactivating protein (RIP), isolated from the seeds of Phytolacca dioica L. PD-S2 has 265 amino-acid residues, and a molecular mass of 29586 Da. The polypeptide chain contains four amino-acid residues more than PAP-S, a type-I RIP isolated from the seeds of the taxonomically related plant Phytolacca americana L. We have compared the amino-acid sequence of PD-S2 with those of two other RIPs with known three-dimensional structure: PAP-S and ricin A-chain (RTA), the active chain of the best known type-2 RIP. This analysis shows an identity of 76% and 33% with PAP-S and RTA respectively, and a similarity of 82% and 54%. Comparison with the PAP sequence, isolated from leaves of P. americana, shows an even higher identity (80%) and similarity (87%). Furthermore, the amino-acid residues reported in other RIPs to be invariant and participate in the definition of the active site (Tyr-76, Tyr-127, Glu-179, Arg-182 and Trp-211; PD-S2 numbering) are all present. Asn-74, Arg-138, Gln-175, and Glu-208 are also conserved, while Asn-209 is substituted by Glu, all residues located in the active-site cleft of RIPs (Tahirov, T.H., Lu, T.-H., Liaw, Y.-C., Chen, J.L. and Lin, J.Y. (1995) Crystal structure of abrin-a at 2.14 A, J. Mol. Biol. 250, 354-367). The polypeptide chain of PD-S2 contains two N-glycosylation sites at Asn-112 and Asn-120, the second of which appears to be linked to sugars. Like PAP-S, PD-S2 does not contain free sulfhydryl groups. The four cysteinyl residues of the two proteins have corresponding sequence positions, most likely with identical S-S pairing.

A low-molecular-mass zinc-binding protein was purified from the eggs of the sea urchin Paracentrotus lividus using procedures that included gel-permeation and anion-exchange chromatography followed by HPLC. The primary structure of this protein was derived from the sequences of peptide fragments obtained by digestion with trypsin and thermolysin. The reconstructed sequence showed the presence of 20 cysteinyl residues, thus resembling that of a metallothionein. The Paracentrotus protein was most similar to the metallothionein of Strongylocentrotus purpuratus, another member of the order of Echinoida, living along the coast of the Pacific Ocean. However, the presence of non-conservative amino acid substitution, together with a deletion of two residues in the Strongylocentrotus metallothionein, make the similarity scores of the two sea urchin proteins lower than that of metallothioneins from vertebrates of the same order. In addition, the present data show that sea urchin metallothioneins display no homology with metallothioneins of any other species.

From the seeds of the Caryophyllaceae Saponaria ocymoides and Vaccaria pyramidata two proteins were purified which have the properties of the type-1 (single-chain) ribosome-inactivating proteins [reviewed by Barbieri, L., Battelli, M. G. & Stirpe, F. (1993) Ribosome-inactivating proteins from plants, Biochim. Biophys. Acta 1154, 237-282]. The proteins have molecular masses of 30.2 kDa (S. ocymoides) and 28.0 kDa (V. pyramidata) and pI greater than 9.5, their N-terminal amino acid sequences are similar to those of saporin-S6 and dianthin 30, ribosome-inactivating proteins from other Caryophyllaceae, and they partially cross-react with sera against these proteins. Both proteins inhibit protein synthesis by a rabbit-reticulocyte lysate with IC50 (concentrations giving 50% inhibition) below 10(-10) M, have a smaller effect on poly(U)-directed phenylalanine polymerisation by rat liver ribosomes (nanomolar IC50, approximately) and on protein synthesis by various cell lines (IC50 ranging from 4 nM to > 3000 nM) and possess rRNA N-glycosidase activity, releasing 1 mol adenine/ribosome.