CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays.

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.

Exome Sequencing Identified a Splice Site Mutation in FHL1 that Causes Uruguay Syndrome, an X-Linked Disorder With Skeletal Muscle Hypertrophy and Premature Cardiac Death.

BACKGROUND: Previously, we reported a rare X-linked disorder, Uruguay syndrome in a single family. The main features are pugilistic facies, skeletal deformities, and muscular hypertrophy despite a lack of exercise and cardiac ventricular hypertrophy leading to premature death. METHODS AND RESULTS: An ≈19 Mb critical region on X chromosome was identified through identity-by-descent analysis of 3 affected males. Exome sequencing was conducted on one affected male to identify the disease-causing gene and variant. A splice site variant (c.502-2A>G) in the FHL1 gene was highly suspicious among other candidate genes and variants. FHL1A is the predominant isoform of FHL1 in cardiac and skeletal muscle. Sequencing cDNA showed the splice site variant led to skipping of exons 6 of the FHL1A isoform, equivalent to the FHL1C isoform. Targeted analysis showed that this splice site variant cosegregated with disease in the family. Western blot and immunohistochemical analysis of muscle from the proband showed a significant decrease in protein expression of FHL1A. Real-time polymerase chain reaction analysis of different isoforms of FHL1 demonstrated that the FHL1C is markedly increased. CONCLUSIONS: Mutations in the FHL1 gene have been reported in disorders with skeletal and cardiac myopathy but none has the skeletal or facial phenotype seen in patients with Uruguay syndrome. Our data suggest that a novel FHL1 splice site variant results in the absence of FHL1A and the abundance of FHL1C, which may contribute to the complex and severe phenotype. Mutation screening of the FHL1 gene should be considered for patients with uncharacterized myopathies and cardiomyopathies.

A novel single base pair duplication in WDR62 causes primary microcephaly.

BACKGROUND: Primary microcephaly is a disorder of the brain resulting in a reduced head circumference that can come along with intellectual disability but with hardly any other neurological abnormalities. CASE PRESENTATION: In this study we report on three Pakistani males from a consanguineous family with 2, 4 and 25 years, diagnosed with autosomal recessive primary microcephaly. By genotyping, Sanger sequencing and using bioinformatical approaches the disease causing mutation was identified and evaluated. CONCLUSION: By using a 250K SNP array, we were able to detect an 11Mb large autozygous region in the MCPH2 locus on chromosome 19q13.12. Sequencing of the associated gene, WDR62, revealed the frameshift causing single base pair duplication, c.2527dupG. This mutation is predicted to affect the structural features of WDR62 which in turn changes the conformation and function of the protein. Aspartic acid (D) at position 843 was found to be conserved among various ortholog species. The present findings will be helpful in genetic diagnosis of patients and future studies of WDR62.

A missense mutation in the PISA domain of HsSAS-6 causes autosomal recessive primary microcephaly in a large consanguineous Pakistani family.

Asymmetric cell division is essential for normal human brain development. Mutations in several genes encoding centrosomal proteins that participate in accurate cell division have been reported to cause autosomal recessive primary microcephaly (MCPH). By homozygosity mapping including three affected individuals from a consanguineous MCPH family from Pakistan, we delineated a critical region of 18.53 Mb on Chromosome 1p21.3-1p13.1. This region contains the gene encoding HsSAS-6, a centrosomal protein primordial for seeding the formation of new centrioles during the cell cycle. Both next-generation and Sanger sequencing revealed a homozygous c.185T>C missense mutation in the HsSAS-6 gene, resulting in a p.Ile62Thr substitution within a highly conserved region of the PISA domain of HsSAS-6. This variant is neither present in any single-nucleotide polymorphism or exome sequencing databases nor in a Pakistani control cohort. Experiments in tissue culture cells revealed that the Ile62Thr mutant of HsSAS-6 is substantially less efficient than the wild-type protein in sustaining centriole formation. Together, our findings demonstrate a dramatic impact of the mutation p.Ile62Thr on HsSAS-6 function and add this component to the list of genes mutated in primary microcephaly.

Development of renal renin-expressing cells does not involve PDGF-B-PDGFR-ß signaling.

Apart from their endocrine functions renin-expressing cells play an important functional role as mural cells of the developing preglomerular arteriolar vessel tree in the kidney. The recruitment of renin-expressing cells from the mesenchyme to the vessel wall is not well understood. Assuming that it may follow more general lines of pericyte recruitment to endothelial tubes we have now investigated the relevance of the platelet-derived growth factor (PDGF)-B-PDGFR-ß signaling pathway in this context. We studied renin expression in kidneys lacking PDGFR-ß in these cells and in kidneys with reduced endothelial PDGF-B expression. We found that expression of renin in the kidneys under normal and stimulated conditions was not different from wild-type kidneys. As expected, PDGFR-ß immunoreactivity was found in mesangial, adventitial and tubulo-interstitial cells but not in renin-expressing cells. These findings suggest that the PDGF-B-PDGFR-ß signaling pathway is not essential for the recruitment of renin-expressing cells to preglomerular vessel walls in the kidney.

Germline mutations in the DNA damage response genes BRCA1, BRCA2, BARD1 and TP53 in patients with therapy related myeloid neoplasms.

BACKGROUND: Therapy related myeloid neoplasms (t-MNs) are complex diseases originating from an interplay between exogenous toxicities and a susceptible organism. It has been hypothesised that in a subset of cases t-MNs develop in the context of hereditary cancer predisposition syndromes. METHODS: The study systematically evaluated pedigrees of patients with t-MNs for cancer incidences and the possibility of a hereditary cancer predisposition syndrome. In addition, mutational analyses were performed using constitutional DNA from index patients, and deleterious heterozygous germline mutations were assessed for loss of heterozygosity in sorted leukaemic cells by single nucleotide polymorphism array. RESULTS: A nuclear pedigree was obtained in 51/53 patients with t-MNs resulting in a total of 828 individuals analysed. With a standardised incidence ratio of 1.03 (95% CI 0.74 to 1.39), the tumour incidence of first- degree relatives was not increased. However, six pedigrees were suggestive for a hereditary breast and ovarian cancer syndrome, three of a Li-Fraumeni like syndrome, and three index patients showed multiple primary neoplasms. Mutational analysis revealed two BRCA1 (c.3112G→T, c.5251C→T), one BRCA2 (c.4027A→G), two BARD1 (C557S) and four TP53 germline mutations (g.18508_18761delinsGCC, c.847C→T, c.845_848dupGGCG, c.1146delA) in nine of 53 (17%) index patients with t-MNs. Loss of heterozygosity in leukaemic cells was demonstrated for the BRCA1c.3112G→T and TP53c.845_848dupGGCG mutations, respectively. CONCLUSION: It is concluded that a proportion of patients with t-MNs carry cancer susceptibility mutations which are likely to contribute to therapy related leukaemogenesis.

Mutation in NSUN2, which encodes an RNA methyltransferase, causes autosomal-recessive intellectual disability.

Causes of autosomal-recessive intellectual disability (ID) have, until very recently, been under researched because of the high degree of genetic heterogeneity. However, now that genome-wide approaches can be applied to single multiplex consanguineous families, the identification of genes harboring disease-causing mutations by autozygosity mapping is expanding rapidly. Here, we have mapped a disease locus in a consanguineous Pakistani family affected by ID and distal myopathy. We genotyped family members on genome-wide SNP microarrays and used the data to determine a single 2.5 Mb homozygosity-by-descent (HBD) locus in region 5p15.32-p15.31; we identified the missense change c.2035G>A (p.Gly679Arg) at a conserved residue within NSUN2. This gene encodes a methyltransferase that catalyzes formation of 5-methylcytosine at C34 of tRNA-leu(CAA) and plays a role in spindle assembly during mitosis as well as chromosome segregation. In mouse brains, we show that NSUN2 localizes to the nucleolus of Purkinje cells in the cerebellum. The effects of the mutation were confirmed by the transfection of wild-type and mutant constructs into cells and subsequent immunohistochemistry. We show that mutation to arginine at this residue causes NSUN2 to fail to localize within the nucleolus. The ID combined with a unique profile of comorbid features presented here makes this an important genetic discovery, and the involvement of NSUN2 highlights the role of RNA methyltransferase in human neurocognitive development.