Expanding the phenotype of biallelic loss-of-function variants in the NSUN2 gene: description of four individuals with juvenile cataract, chronic nephritis, or brain anomaly as novel complications

The NSUN2 gene encodes a tRNA cytosine methyltransferase that functions in the maturation of leucyl tRNA (Leu) (CAA) precursors, which is crucial for the anticodon-codon pairing and correct translation of mRNA. Biallelic loss of function variants in NSUN2 are known to cause moderate to severe intellectual disability. Microcephaly, postnatal growth retardation, and dysmorphic facial features are common complications in this genetic disorder, and delayed puberty is occasionally observed. Here, we report four individuals, two sets of siblings, with biallelic loss-of-function variants in the NSUN2 gene. The first set of siblings have compound heterozygous frameshift variants: c.546_547insCT, p.Met183Leufs*13; c.1583del, p.Pro528Hisfs*19, and the other siblings carry a homozygous frameshift variant: c.1269dup, p.Val424Cysfs*14. In addition to previously reported clinical features, the first set of siblings showed novel complications of juvenile cataract and chronic nephritis. The other siblings showed hypomyelination and simplified gyral pattern in neuroimaging. NSUN2-related intellectual disability is a very rare condition, and less than 20 cases have been reported previously. Juvenile cataract, chronic nephritis, and brain anomaly shown in the present patients have not been previously described. Our report suggests clinical diversity of NSUN2-related intellectual disability.</p

Comprehensive investigation of <i>CASK</i> mutations and other genetic etiologies in 41 patients with intellectual disability and microcephaly with pontine and cerebellar hypoplasia (MICPCH)

<div><p>The <i>CASK</i> gene (Xp11.4) is highly expressed in the mammalian nervous system and plays several roles in neural development and synaptic function. Loss-of-function mutations of <i>CASK</i> are associated with intellectual disability and microcephaly with pontine and cerebellar hypoplasia (MICPCH), especially in females. Here, we present a comprehensive investigation of 41 MICPCH patients, analyzed by mutational search of <i>CASK</i> and screening of candidate genes using an SNP array, targeted resequencing and whole-exome sequencing (WES). In total, we identified causative or candidate genomic aberrations in 37 of the 41 cases (90.2%). <i>CASK</i> aberrations including a rare mosaic mutation in a male patient, were found in 32 cases, and a mutation in <i>ITPR1</i>, another known gene in which mutations are causative for MICPCH, was found in one case. We also found aberrations involving genes other than <i>CASK</i>, such as <i>HDAC2</i>, <i>MARCKS</i>, and possibly <i>HS3ST5</i>, which may be associated with MICPCH. Moreover, the targeted resequencing screening detected heterozygous variants in <i>RELN</i> in two cases, of uncertain pathogenicity, and WES analysis suggested that concurrent mutations of both <i>DYNC1H1</i> and <i>DCTN1</i> in one case could lead to MICPCH. Our results not only identified the etiology of MICPCH in nearly all the investigated patients but also suggest that MICPCH is a genetically heterogeneous condition, in which <i>CASK</i> inactivating mutations appear to account for the majority of cases.</p></div

Candidate genes for the target resequencing.

<p>Candidate genes for the target resequencing.</p

Schemes of the point mutations and CNVs involving CASK.

<p>(A) Schematic representation of the structure of <i>CASK</i> domains (NCBI Reference Sequence: NP_003679.2) and the position of the point mutations in patients 1–23. CaMK: calmodulin-dependent kinase, L27: LIN-2 and LIN-7 interaction, PDZ: PSD-95-Dlg-ZO1, SH3: Src homologous 3, GK: guanylate kinase. (B) Mapping of the CNVs involving <i>CASK</i> identified in patients 24–32. Black horizontal bars indicate the deletions and gray bars indicate the duplications, respectively, and horizontal arrows indicate genes and their directions. Dashed lines enlarge around <i>CASK</i>. The regional information is from the UCSC built on February 2009 (GRCh37/hg19).</p

Detailed analysis of the mosaicism of <i>CASK</i> in patient 23.

<p><b>A</b> Sequence chromatogram showing a heterozygous-like pattern in the latter part of exon 15 (arrow). <b>B</b> Scheme of the indel mutation. Compared with the reference allele (upper), the affected allele (lower) had a 21-bp deletion and a 2-bp insertion at the exon-intron junction of exon 15 and intron 15. <b>C</b> Results of the genomic PCR using WT-specific and indel-specific primer sets in the patient and a male control. The red box indicates a product amplified only in the patient with the indel-specific primer sets. M: marker; phiX174 RF DNA/Hae III Fragments, P: patient 23, C: control, N: negative control, no DNA added. <b>D</b> Real-time quantitative PCR of genomic DNA from patient 23 and male and female controls. While the relative copy number of the male control is naturally approximately half of that of the female control, those amplified with both WT-specific and deletion-specific primers in the patient are also approximately half of that of the male control.</p

Genomic analysis of candidate genes other than <i>CASK</i>.

<p>(A) Result of the SNP array in patient 33 showing Heterozygous deletion at 6q21-q22.31 including <i>HDAC2</i> and <i>MARCKS</i>. This result is described as follows: arr 6q21q22.31(109,497,085–122,505,593)x1. The double-headed arrow indicates the deletion. (B) Mapping of the heterozygous deletion in patient 33. The red box denotes <i>HDAC2</i> and <i>MARCKS</i>. (C) Electropherograms depicting the mutations of <i>RELN</i> detected by targeted resequencing. Arrows indicate the mutated nucleotides. (upper) c.4918A>G (p.I1640V) in patient 34, (lower) c.7093G>A (p.V2365M) in patient 35. (D) Conservation of amino acids around each mutation of <i>RELN</i> in patient 34 (upper) and patient 35 (lower). The red box denotes the amino acid substituted by the mutation. (E) Electropherograms depicting the mutations detected by whole exome sequencing. Each arrow indicates the mutated nucleotide. (upper) c.1677dupG (p.R560Afs*20) of <i>CASK</i> in patient 11, (lower) c.7753A>C (p.T2585P) of <i>ITPR1</i> in patient 36. (F) Electropherograms depicting the mutations of <i>DYNC1H1</i> and <i>DCTN1</i> in patient 37 and her parents. Arrows indicate the mutated nucleotides. The left three panels indicating c.11824C>T (p.P3942S) of <i>DYNC1H1</i> show that the mutation is inherited from the mother, and the right three panels indicating c.497C>G (p.S166C) of <i>DCTN1</i> show the mutation is inherited from the father.</p

Molecular and Clinical Studies in 138 Japanese Patients with Silver-Russell Syndrome

<div><p>Background</p><p>Recent studies have revealed relative frequency and characteristic phenotype of two major causative factors for Silver-Russell syndrome (SRS), i.e. epimutation of the <i>H19</i>-differentially methylated region (DMR) and uniparental maternal disomy 7 (upd(7)mat), as well as multilocus methylation abnormalities and positive correlation between methylation index and body and placental sizes in <i>H19</i>-DMR epimutation. Furthermore, rare genomic alterations have been found in a few of patients with idiopathic SRS. Here, we performed molecular and clinical findings in 138 Japanese SRS patients, and examined these matters.</p> <p>Methodology/Principal Findings</p><p>We identified <i>H19</i>-DMR epimutation in cases 1–43 (group 1), upd(7)mat in cases 44–52 (group 2), and neither <i>H19</i>-DMR epimutation nor upd(7)mat in cases 53–138 (group 3). Multilocus analysis revealed hyper- or hypomethylated DMRs in 2.4% of examined DMRs in group 1; in particular, an extremely hypomethylated <i>ARHI</i>-DMR was identified in case 13. Oligonucleotide array comparative genomic hybridization identified a ∼3.86 Mb deletion at chromosome 17q24 in case 73. Epigenotype-phenotype analysis revealed that group 1 had more reduced birth length and weight, more preserved birth occipitofrontal circumference (OFC), more frequent body asymmetry and brachydactyly, and less frequent speech delay than group 2. The degree of placental hypoplasia was similar between the two groups. In group 1, the methylation index for the <i>H19</i>-DMR was positively correlated with birth length and weight, present height and weight, and placental weight, but with neither birth nor present OFC.</p> <p>Conclusions/Significance</p><p>The results are grossly consistent with the previously reported data, although the frequency of epimutations is lower in the Japanese SRS patients than in the Western European SRS patients. Furthermore, the results provide useful information regarding placental hypoplasia in SRS, clinical phenotypes of the hypomethylated <i>ARHI</i>-DMR, and underlying causative factors for idiopathic SRS.</p> </div

Oligonucleotide array CGH in case 73, showing a ∼3.86 Mb deletion at chromosome 17q24.

<p>The black, the red, and the green dots denote signals indicative of the normal, the increased(>+0.5), and the decreased (< –1.0) copy numbers, respectively. The horizontal bar with arrowheads indicates a ∼2.3 Mb deletion identified in a patient with Carney complex and SRS-like phenotype <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060105#pone.0060105-Blyth1" target="_blank">[44]</a>, and the black square represent a ∼65 kb segment harboring the breakpoint of a <i>de novo</i> translocation 46,XY,t(1;17)(q24;q23–q24) identified in a patient with SRS phenotype <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060105#pone.0060105-Midro1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060105#pone.0060105-Drr1" target="_blank">[46]</a>.</p

Correlation analyses in patients with <i>H19</i>-DMR hypomethylations.

<p>SDS: standard deviation score; and OFC: occipitofrontal circumference.</p>*<p>The mean value of MIs for CG5, CG6, CG7, and CG9 obtained by pyrosequencing analysis.</p><p>Significant <i>P</i>-values(<0.05) are boldfaced.</p

Analysis of the <i>ARHI</i>-DMR in case 13.

<p>For bisulfite sequencing, each line indicates a single clone, and each circle denotes a CpG dinucleotide; the cytosine residues at the CpG dinucleotides are usually unmethylated after paternal transmission (open circles) and methylated after maternal transmission (filled circles). Electrochromatograms delineate the sequences of the primer binding sites utilized for the methylation analysis.</p