Novel IRF6 Mutations Detected in Orofacial Cleft Patients by Targeted Massively Parallel Sequencing.

Common variants in interferon regulatory factor 6 ( IRF6) have been associated with nonsyndromic cleft lip with or without cleft palate (NSCL/P) as well as with tooth agenesis (TA). These variants contribute a small risk towards the 2 congenital conditions and explain only a small percentage of heritability. On the other hand, many IRF6 mutations are known to be a monogenic cause of disease for syndromic orofacial clefting (OFC). We hypothesize that IRF6 mutations in some rare instances could also cause nonsyndromic OFC. To find novel rare variants in IRF6 responsible for nonsyndromic OFC and TA, we performed targeted multiplex sequencing using molecular inversion probes (MIPs) in 1,072 OFC patients, 67 TA patients, and 706 controls. We identified 3 potentially pathogenic de novo mutations in OFC patients. In addition, 3 rare missense variants were identified, for which pathogenicity could not unequivocally be shown, as all variants were either inherited from an unaffected parent or the parental DNA was not available. Retrospective investigation of the patients with these variants revealed the presence of lip pits in one of the patients with a de novo mutation suggesting a Van der Woude syndrome (VWS) phenotype, whereas, in other patients, no lip pits were identified.

Germline activating TYK2 mutations in pediatric patients with two primary acute lymphoblastic leukemia occurrences.

The contribution of genetic predisposing factors to the development of pediatric acute lymphoblastic leukemia (ALL), the most frequently diagnosed cancer in childhood, has not been fully elucidated. Children presenting with multiple de novo leukemias are more likely to suffer from genetic predisposition. Here, we selected five of these patients and analyzed the mutational spectrum of normal and malignant tissues. In two patients, we identified germline mutations in TYK2, a member of the JAK tyrosine kinase family. These mutations were located in two adjacent codons of the pseudokinase domain (p.Pro760Leu and p.Gly761Val). In silico modeling revealed that both mutations affect the conformation of this autoregulatory domain. Consistent with this notion, both germline mutations promote TYK2 autophosphorylation and activate downstream STAT family members, which could be blocked with the JAK kinase inhibitor I. These data indicate that germline activating TYK2 mutations predispose to the development of ALL.

De novo WNT5A-associated autosomal dominant Robinow syndrome suggests specificity of genotype and phenotype.

Robinow Syndrome (RS), a rare skeletal dysplasia syndrome, is characterized by dysmorphic features resembling a fetal face, mesomelic limb shortening, hypoplastic external genitalia in males, and renal and vertebral anomalies. Both autosomal dominant and autosomal recessive patterns of inheritance have been reported. Since the description of autosomal dominant Robinow Syndrome (ADRS; OMIM 180700) in 1969 by Meinhard Robinow and colleagues, the molecular etiology remained elusive until only recently. WNT5A was proposed to be the candidate gene for ADRS, as mutations were found in two affected families, one of those being the originally described index family. We report three families with RS caused by novel heterozygous WNT5A mutations, which were confirmed in the first family by whole exome sequencing, and in all by Sanger sequencing. To our knowledge, this is the largest number of published families with ADRS in whom a WNT5A mutation was identified. Families 1 and 2 are the first cases showing de novo inheritance in the affected family members and thus strengthen the evidence for WNT5A as the causative gene in ADRS. Finally, we propose WNT5A mutation specificity in ADRS, which may affect interactions with other proteins in the Wnt pathway.

Genotype and clinical care correlations in craniosynostosis: findings from a cohort of 630 Australian and New Zealand patients.

Craniosynostosis is one of the most common craniofacial disorders encountered in clinical genetics practice, with an overall incidence of 1 in 2,500. Between 30% and 70% of syndromic craniosynostoses are caused by mutations in hotspots in the fibroblast growth factor receptor (FGFR) genes or in the TWIST1 gene with the difference in detection rates likely to be related to different study populations within craniofacial centers. Here we present results from molecular testing of an Australia and New Zealand cohort of 630 individuals with a diagnosis of craniosynostosis. Data were obtained by Sanger sequencing of FGFR1, FGFR2, and FGFR3 hotspot exons and the TWIST1 gene, as well as copy number detection of TWIST1. Of the 630 probands, there were 231 who had one of 80 distinct mutations (36%). Among the 80 mutations, 17 novel sequence variants were detected in three of the four genes screened. In addition to the proband cohort there were 96 individuals who underwent predictive or prenatal testing as part of family studies. Dysmorphic features consistent with the known FGFR1-3/TWIST1-associated syndromes were predictive for mutation detection. We also show a statistically significant association between splice site mutations in FGFR2 and a clinical diagnosis of Pfeiffer syndrome, more severe clinical phenotypes associated with FGFR2 exon 10 versus exon 8 mutations, and more frequent surgical procedures in the presence of a pathogenic mutation. Targeting gene hot spot areas for mutation analysis is a useful strategy to maximize the success of molecular diagnosis for individuals with craniosynostosis.

Secondary and tertiary structure modeling reveals effects of novel mutations in polycystic liver disease genes PRKCSH and SEC63.

Polycystic liver disease (PCLD) is characterized by intralobular bile duct cysts in the liver. It is caused by mutations in PRKCSH, encoding hepatocystin, and SEC63, encoding Sec63p. The main goals of this study were to screen for novel mutations and to analyze mutations for effects on protein structure and function. We screened 464 subjects including 76 probands by direct sequencing or conformation-sensitive capillary electrophoresis. We analyzed the effects of all known and novel mutations using a combination of splice site recognition, evolutionary conservation, secondary and tertiary structure predictions, PolyPhen, and pMut and sift. We identified a total of 26 novel mutations in PRKCSH (n = 14) and SEC63 (n = 12), including four splice site mutations, eight insertions/ deletions, six non-sense mutations, and eight missense mutations. Out of 48 PCLD mutations, 13 were predicted to affect splicing. Most mutations were located in highly conserved regions and homology modeling for two domains of Sec63p showed severe effects of the residue substitutions. In conclusion, we identified 26 novel mutations associated with PCLD and we provide in silico analysis in order to delineate the role of these mutations.

Further clinical and molecular delineation of the 9q subtelomeric deletion syndrome supports a major contribution of EHMT1 haploinsufficiency to the core phenotype.

BACKGROUND: The 9q subtelomeric deletion syndrome (9qSTDS) is clinically characterised by moderate to severe mental retardation, childhood hypotonia and facial dysmorphisms. In addition, congenital heart defects, urogenital defects, epilepsy and behavioural problems are frequently observed. The syndrome can be either caused by a submicroscopic 9q34.3 deletion or by intragenic EHMT1 mutations leading to haploinsufficiency of the EHMT1 gene. So far it has not been established if and to what extent other genes in the 9q34.3 region contribute to the phenotype observed in deletion cases. This study reports the largest cohort of 9qSTDS cases so far. METHODS AND RESULTS: By a multiplex ligation dependent probe amplification (MLPA) approach, the authors identified and characterised 16 novel submicroscopic 9q deletions. Direct sequence analysis of the EHMT1 gene in 24 patients exhibiting the 9qSTD phenotype without such deletion identified six patients with an intragenic EHMT1 mutation. Five of these mutations predict a premature termination codon whereas one mutation gives rise to an amino acid substitution in a conserved domain of the protein. CONCLUSIONS: The data do not provide any evidence for phenotype-genotype correlations between size of the deletions or type of mutations and severity of clinical features. Therefore, the authors confirm the EHMT1 gene to be the major determinant of the 9qSTDS phenotype. Interestingly, five of six patients who had reached adulthood had developed severe psychiatric pathology, which may indicate that EHMT1 haploinsufficiency is associated with neurodegeneration in addition to neurodevelopmental defect.