Jon Warner

The ASP1 gene of Saccharomyces cerevisiae, encoding the intracellular isozyme of l-asparaginase

Saccharomyces cerevisiae produces two L-asparaginases (ASPs), intracellular ASP I and cell-wall ASP II. In this report, the ASP-I-encoding gene, ASP1, has been identified by homology cloning based on the structures of ASPs from other organisms. Its deduced protein product has a subunit M(r) of 41,414, and shows substantial sequence homology to the bacterial amidohydrolase family. The product of the S. cerevisiae ASP3 gene, a further member of this family, encoding the nitrogen catabolite-regulated cell-wall ASP II, has 46% overall sequence identity to ASP1. Duplication of ancestral asparaginase genes, resulting in separate intra- and extracellular isozymes, appears to have occurred independently in the prokaryotic and eukaryotic lineages. Exact physical mapping of the new cloned ASP1 gene locates it 73% of the distance from the left telomere of chromosome IV, at a position precisely matching the known genetic map location of ASP1. This, along with the structural features of the clone, confirms that ASP1 is the structural gene encoding cytoplasmic ASP I in S. cerevisiae. Sequence analysis of the ethylmethanesulfonate-induced asp1-12 allele of strain XE101-1A revealed a C-->T transition altering Ala176 to Val. This residue lies within a highly conserved region, and the results suggests a critical function for Ala176 in ASP function. Expression of ASP1 and other recombinant ASPs may allow access to improved products for use in the chemotherapy of leukaemia.

Targeted sequencing of the Paget's disease associated 14q32 locus identifies several missense coding variants in RIN3 that predispose to Paget's disease of bone

Pagets disease of bone (PDB) is a common disorder with a strong genetic component characterized by increased but disorganized bone remodelling. Previous genome-wide association studies identified a locus on chromosome 14q32 tagged by rs10498635 which was significantly associated with susceptibility to PDB in several European populations. Here we conducted fine-mapping and targeted sequencing of the candidate locus to identify possible functional variants. Imputation in 741 PDB patients and 2699 controls confirmed that the association was confined to a 60 kb region in the RIN3 gene and conditional analysis adjusting for rs10498635 identified no new independent signals. Sequencing of the RIN3 gene identified a common missense variant (p.R279C) that was strongly associated with the disease (OR = 0.64; P = 1.4 × 10−9), and was in strong linkage disequilibrium with rs10498635. A further 13 rare missense variants were identified, seven of which were novel and detected only in PDB cases. When combined, these rare variants were over-represented in cases compared with controls (OR = 3.72; P = 8.9 × 10−10). Most rare variants were located in a region that encodes a proline-rich, intrinsically disordered domain of the protein and many were predicted to be pathogenic. RIN3 was expressed in bone tissue and its expression level was ∼10-fold higher in osteoclasts compared with osteoblasts. We conclude that susceptibility to PDB at the 14q32 locus is mediated by a combination of common and rare coding variants in RIN3 and suggest that RIN3 may contribute to PDB susceptibility by affecting osteoclast function.

Analysis of the genomic organisation of a small chromosome of Leishmania braziliensis M2903 reveals two genes encoding GTP-binding proteins, one of which belongs to a new G-protein family and is an antigen

Leishmania braziliensis M2903 contains a highly amplified small chromosome. This work is aimed at resolving its structural organization and determining whether this unusual chromosome contains specific genes encoding proteins with important functions in disease pathology or drug resistance. Our results show that the M2903 250-kb small chromosome contains LD1 sequences and has an inverted repeat structure. The LD1 sequences and two cDNAs (cDNA2 and cDNA53) were mapped on a cosmid contig, and the two cDNAs and the corresponding genomic fragments from the small chromosome were sequenced. The gene encoding cDNA2 predicts a putative GTP-binding protein with homology to other GTP-binding proteins only in the G-1 domain region; however, four other conserved motifs can be recognized. Sequence similarity to cDNA53 is located in at least five chromosomes, and its small chromosome copy is a pseudogene. An open reading frame downstream of the cDNA53 pseudogene predicts another GTP-binding protein that belongs to a new G-protein family with an unusual conserved GTP-binding domain and a newly characterized conserved sequence motif. A portion of this GTP-binding protein gene was studied previously in L. aethiopica as a recombinant antigen that reacts with human antibodies.

Genetic epidemiology of motor neuron disease-associated variants in the Scottish population

Genetic understanding of motor neuron disease (MND) has evolved greatly in the past 10 years, including the recent identification of association between MND and variants in TBK1 and NEK1. Our aim was to determine the frequency of pathogenic variants in known MND genes and to assess whether variants in TBK1 and NEK1 contribute to the burden of MND in the Scottish population. SOD1, TARDBP, OPTN, TBK1, and NEK1 were sequenced in 441 cases and 400 controls. In addition to 44 cases known to carry a C9orf72 hexanucleotide repeat expansion, we identified 31 cases and 2 controls that carried a loss-of-function or pathogenic variant. Loss-of-function variants were found in TBK1 in 3 cases and no controls and, separately, in NEK1 in 3 cases and no controls. This study provides an accurate description of the genetic epidemiology of MND in Scotland and provides support for the contribution of both TBK1 and NEK1 to MND susceptibility in the Scottish population.

Improved PCR based methods for detecting C9orf72 hexanucleotide repeat expansions.

Due to the GC-rich, repetitive nature of C9orf72 hexanucleotide repeat expansions, PCR based detection methods are challenging. Several limitations of PCR have been reported and overcoming these could help to define the pathogenic range. There is also a need to develop improved repeat-primed PCR assays which allow detection even in the presence of genomic variation around the repeat region. We have optimised PCR conditions for the C9orf72 hexanucleotide repeat expansion, using betaine as a co-solvent and specific cycling conditions, including slow ramping and a high denaturation temperature. We have developed a flanking assay, and repeat-primed PCR assays for both 3′ and 5′ ends of the repeat expansion, which when used together provide a robust strategy for detecting the presence or absence of expansions greater than ∼100 repeats, even in the presence of genomic variability at the 3′ end of the repeat. Using our assays, we have detected repeat expansions in 47/442 Scottish ALS patients. Furthermore, we recommend the combined use of these assays in a clinical diagnostic setting.

Semi-dominant X-chromosome linked learning disability with progressive ataxia, spasticity and dystonia associated with the novel MECP2 variant p.V122A: Akin to the new MECP2 duplication syndrome?

A novel X-chromosome linked phenotype is reported. Three affected males had learning disability in early childhood and subsequently developed progressive ataxia, dystonia, and spasticity with death at ages 9, 14 and 19 years. Two female obligate carriers had learning difficulties with psychosis in one case. A third, possible carrier had learning difficulties and epilepsy. A family study indicates that this inherited syndrome is most likely due to an unreported MECP2 variant, p.V122A, located in the methyl binding domain of the MECP2 protein. The clinical features are similar to those present in the newly reported MECP2 duplication syndrome. Non-progressive neuropsychiatric symptoms in female relatives of a male child with learning disability, ataxia and progressive spasticity may constitute a clue to inherited, MECP2 pathogenesis.

De novo mutations in autosomal recessive congenital malformations.

To the Editor: In suspected autosomal recessive disease without a genetic diagnosis, the risk to future pregnancies is generally reported as 25%, assuming inheritance of one causative allele from each parent. However, it is also possible that a de novo mutation (DNM) could contribute to disease in these cases. The contribution of DNMs to autosomal dominant developmental disorders is widely recognized, but their frequency in autosomal recessive disease is thought to be low, as found in a recent Genetics in Medicine article by Retterer et al.,1 titled “Clinical Application of Whole-Exome Sequencing Across Clinical Indications,” and other studies.2,3 The genetic diagnosis of such conditions is important because it allows precise understanding of disease etiology and accurate reporting of recurrence risk. This enables families to make informed decisions about future reproduction and provides the opportunity for earlier genetic screening in future pregnancies. Without a genetic diagnosis, recurrence risk is estimated on the basis of the clinical phenotype alone. In a series of nine families in which severe fetal malformation led to the termination of a pregnancy, we used trio-based exome sequencing to identify two unrelated cases in which an autosomal recessive disorder was caused by the combination of one de novo and one inherited variant. Compound heterozygous loss-offunction variants in EVC2 were identified in the first fetus. Variants in this gene have been shown to cause Ellis-van Creveld syndrome, which was consistent with the phenotype of the fetus. The first variant was a 1-bp deletion in exon 16 (ENST00000344408.5:c.2746delA) that results in a frameshift generating a premature stop codon (ENSP00000342144.5: p.Ser916AlafsTer6). This was inherited from the mother. The second was a de novo nonsense variant in exon 18 (ENST00000344408.5:c.3141G>A; ENSP00000342144.5:p. Trp1047Ter), which allele-specific polymerase chain reaction confirmed had arisen on the paternal haplotype. In the second family, compound heterozygous loss-of-function variants in FRAS1 were identified in the fetus; these mutations cause Fraser syndrome, which was consistent with the phenotype. The first variant was a 20-bp deletion in exon 41 (ENST 00000264895.6:c.5664_5665+19delinsT), leading to the loss of two bases of coding sequence and a donor splice site, which is predicted to lead to skipping of exon 41 and a frameshift in the open reading frame, generating a premature stop codon (ENST00000264895.6:p.Asp1845ThrfsTer10). This variant was inherited from the mother. The second variant was a de novo 1-bp deletion in exon 66 (ENST00000264895.6:c.10287delC), resulting in a frameshift mutation and a premature stop codon (ENSP00000264895.6:p.Tyr3429Ter). Allele-specific polymerase chain reaction confirmed that the DNM had arisen on the paternal chromosome. The variants were validated by Sanger sequencing, and the biological relationships within both trios were confirmed, showing that these were true DNMs. For these two fetuses, the clinical phenotypes were suggestive of autosomal recessive syndromes with a reported recurrence risk of 25%. In both cases, because one of the causative variants arose de novo, the recurrence risk is markedly reduced, with significant implications for future reproductive decisions. A small finite risk remains, however, owing to the possibility of germ-line mosaicism in the father in both families. Three recently published trio-based exome sequencing studies, each including several thousand cases, a proportion of which presented as congenital malformations, did not report a single case of recessive disorder in which one of the causative alleles was a DNM.1–3 Retterer et al.1 reported exome sequencing in more than 3,000 cases and Yang et al.2 in more than 2,000 cases; together these studies report more than 400 autosomal recessive molecular diagnoses, without a single case of a DNM contributing to the genetic etiology. The Deciphering Developmental Disorders project did not identify enrichment for any functional class of DNMs in autosomal recessive developmental disorder–linked genes.3 Although our study size is small, it suggests that DNMs may have a more important role in autosomal recessive congenital malformation than has previously been considered. Given that the identification of a causative DNM in recessive disease leads to a low predicted recurrence risk but has hitherto been infrequently reported, we recommend that the frequency of this mode of inheritance be analyzed systematically in a larger series of cases, particularly in patients presenting with a congenital malformation. Future studies of recessive disease should specifically report whether this mechanism contributes to any of their cases. For accurate clinical reporting, this mode of inheritance should always be considered a possible contributor to the cause of autosomal recessive congenital malformation. Clinical confirmation of parental carrier status will help to ensure accurate reporting of recurrence risk in autosomal recessive disease.

Corrigendum to “Genetic epidemiology of motor neuron disease-associated variants in the Scottish population.” [Neurobiol. Aging 51 (2017) 178.e11–178.e20]

“The authors thank the patients for consenting to research. In addition, they acknowledge Alona Sosinsky and the Imperial College BRC Genomics facility for bioinformatics support. They acknowledge Generation Scotland for providing control samples and thank Shona Kerr and Archie Campbell for assistance in provision of Generation Scotland samples. The authors acknowledge Cat Graham for providing statistical guidance and also thank David Parry and Sophie Marion de Proce for providing helpful comments on the manuscript.

PolyQ Tract Toxicity in SCA1 is Length Dependent in the Absence of CAG Repeat Interruption

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by an expansion of a polyglutamine tract within the ATXN1 gene. Normal alleles have been reported to range from 6 to 35 repeats, intermediate alleles from 36 to 38 repeats and fully penetrant pathogenic alleles have at least 39 repeats. This distribution was based on relatively few samples and the narrow intermediate range makes the accuracy of the repeat sizing crucial for interpreting and reporting diagnostic tests, which can vary between laboratories. Here, we examine the distribution of 6378 SCA1 chromosomes and identify a very late onset SCA1 family with a fully penetrant uninterrupted pathogenic allele containing 38 repeats. This finding supports the theory that polyQ toxicity is related to the increase of the length of the inherited tracts and not as previously hypothesized to the structural transition occurring above a specific threshold. In addition, the threshold of toxicity shifts to a shorter polyQ length with the increase of the lifespan in SCA1. Furthermore, we show that SCA1 intermediate alleles have a different behavior compared to the other polyglutamine disorders as they do not show reduced penetrance when uninterrupted. Therefore, the pathogenic mechanism in SCA1 is distinct from other cytosine-adenine-guanine (CAG) repeat disorders. Accurately sizing repeats is paramount in precision medicine and can be challenging particularly with borderline alleles. We examined plasmids containing cloned CAG repeat tracts alongside a triplet repeat primed polymerase chain reaction (TP PCR) CAG repeat ladder to improve accuracy in repeat sizing by fragment analysis. This method accurately sizes the repeats irrespective of repeat composition or length. We also improved the model for calculating repeat length from fragment analysis sizing by fragment analyzing 100 cloned repeats of known size. Therefore, we recommend these methods for accurately sizing repeat lengths and restriction enzyme digestion to identify interruptions for interpretation of a given allele’s pathogenicity.

De novo repeat interruptions are associated with reduced somatic instability and mild or absent clinical features in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is a multisystem disorder, caused by expansion of a CTG trinucleotide repeat in the 3′-untranslated region of the DMPK gene. The repeat expansion is somatically unstable and tends to increase in length with time, contributing to disease progression. In some individuals, the repeat array is interrupted by variant repeats such as CCG and CGG, stabilising the expansion and often leading to milder symptoms. We have characterised three families, each including one person with variant repeats that had arisen de novo on paternal transmission of the repeat expansion. Two individuals were identified for screening due to an unusual result in the laboratory diagnostic test, and the third due to exceptionally mild symptoms. The presence of variant repeats in all three expanded alleles was confirmed by restriction digestion of small pool PCR products, and allele structures were determined by PacBio sequencing. Each was different, but all contained CCG repeats close to the 3′-end of the repeat expansion. All other family members had inherited pure CTG repeats. The variant repeat-containing alleles were more stable in the blood than pure alleles of similar length, which may in part account for the mild symptoms observed in all three individuals. This emphasises the importance of somatic instability as a disease mechanism in DM1. Further, since patients with variant repeats may have unusually mild symptoms, identification of these individuals has important implications for genetic counselling and for patient stratification in DM1 clinical trials.