Asaf Ta-Shma

A deleterious mutation in DNAJC6 encoding the neuronal-specific clathrin-uncoating co-chaperone auxilin, is associated with juvenile parkinsonism.

Parkinson disease is caused by neuronal loss in the substantia nigra which manifests by abnormality of movement, muscle tone, and postural stability. Several genes have been implicated in the pathogenesis of Parkinson disease, but the underlying molecular basis is still unknown for ∼70% of the patients. Using homozygosity mapping and whole exome sequencing we identified a deleterious mutation in DNAJC6 in two patients with juvenile Parkinsonism. The mutation was associated with abnormal transcripts and marked reduced DNAJC6 mRNA level. DNAJC6 encodes the HSP40 Auxilin, a protein which is selectively expressed in neurons and confers specificity to the ATPase activity of its partner Hcs70 in clathrin uncoating. In Auxilin null mice it was previously shown that the abnormally increased retention of assembled clathrin on vesicles and in empty cages leads to impaired synaptic vesicle recycling and perturbed clathrin mediated endocytosis. Endocytosis function, studied by transferring uptake, was normal in fibroblasts from our patients, likely because of the presence of another J-domain containing partner which co-chaperones Hsc70-mediated uncoating activity in non-neuronal cells. The present report underscores the importance of the endocytic/lysosomal pathway in the pathogenesis of Parkinson disease and other forms of Parkinsonism.

CCDC65 mutation causes primary ciliary dyskinesia with normal ultrastructure and hyperkinetic cilia

Background Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating. Methods Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion. Results A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells. Conclusion Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.

A human laterality disorder caused by a homozygous deleterious mutation in MMP21

Background Laterality in the vertebrate embryo is determined by left-right asymmetric gene expression driven by the flow of extraembryonic fluid across the embryonic node. Defects in these processes cause heterotaxy, the abnormal formation and arrangement of visceral organs that can range from complete inversion of symmetry to the selective misarrangement of organs. However, our understanding of the genetic causality for laterality defects in human beings remains relatively limited. Methods We performed whole exome sequencing in a consanguineous family with heterotaxia. To interrogate the pathogenic potential of the discovered variant, we used an in vivo system in which the potential of the candidate gene to induce L-R asymmetry was tested by transient suppression and CRISPR/Cas9-induced deletions. We also used in vitro assays to test a possible link between our exome-derived candidate and Notch signaling. Results We identified a homozygous 2 bp deletion in MMP21, encoding matrix metalloproteinase-21, as the sole coding mutation that segregated with the phenotype. Transient suppression or CRISPR/Cas9-mediated deletion of mmp21 in zebrafish embryos induced cardiac looping defects, with concomitant disruption of laterality markers in the lateral plate mesoderm and disrupted notch signalling in vitro and in vivo. Conclusions Our data implicate loss of MMP21 as a cause of heterotaxy in humans with concomitant defects in Notch signaling. In support of this finding, a homozygous missense mutation in MMP21 was identified previously in mice with N-Ethyl-N-Nitrosourea (ENU)-induced heterotaxy. Taken together, these observations suggest a role of matrix metalloproteinases in the establishment of asymmetric organ development, likely through the regulation of morphogenetic signals.

A human laterality disorder associated with recessive CCDC11 mutation

Background Significant advancements in understanding the molecular pathophysiology of laterality determination were recently made. However, there are large gaps in our knowledge of the initial processes that lead to laterality defects, such as heterotaxy syndrome (HS, also known as situs ambiguous) and situs inversus totalis (SIT). The former refers to abnormal distribution of visceral organs, and the latter refers to a complete laterality inversion of both abdominal and thoracic viscera. Methods In order to identify a mutated gene in SIT and HS patients, the authors performed homozygosity mapping in a consanguineous family with laterality disorders identified in two siblings. Results A homozygous deleterious mutation in the CCDC11 gene was identified in the patients. The mutation resulted in an abnormally smaller protein in the patients skin fibroblasts. The parents and five healthy siblings were heterozygous for the mutation, which was not present in 112 anonymous controls. Conclusions Few genes have been associated with both SIT and HS, usually accompanied by other abnormalities. The authors suggest that CCDC11 is associated with autosomal recessive laterality defects of diverse phenotype resulting in SIT in one individual family member who is otherwise healthy, and in complex laterality anomalies (HS) in another member. This report underscores the importance of CCDC11 in laterality determination.

Conotruncal malformations and absent thymus due to a deleterious NKX2-6 mutation

Background Truncus arteriosus (TA) accounts for ∼1% of congenital heart defects. The aetiology of isolated TA is largely unknown but when occurring as part of a syndrome, it is mostly associated with chromosome 22q11 deletion. Vice versa, the most common congenital heart defects associated with chromosome 22q11 deletion are conotruncal malformations. In this study we investigated the cause of multiple conotruncal malformations accompanied by athymia in a consanguineous family. Methods and results Whole exome analysis revealed a homozygous deleterious mutation in the NKX2-6 gene. Conclusions NKX2-6 encodes a homeobox-containing protein which is expressed in mouse embryo at E8.0-E9.5 at the caudal pharyngeal arches and the outflow tract. A single missense mutation was previously implicated in the aetiology of familial isolated TA; however, null mice are entirely normal. The clear phenotype associated with a homozygous deleterious mutation in the present report, falls well within the spectrum of the cardiac defects seen in DiGeorge syndrome, is in agreement with NKX2-6 downstream location in the TBX1 signalling pathway and confirms NKX2-6 role in human cardiogenesis.

Isolated truncus arteriosus associated with a mutation in the plexin-D1 gene.

Truncus arteriosus accounts for approximately 1% of congenital heart defects and the cause of isolated non‐syndromic truncus arteriosus is largely unknown. In order to identify the underlying molecular defect in a consanguineous family with recurrent tuncus arteriosus, homozygosity mapping followed by whole exome sequencing was performed. This resulted in the identification of a homozygous mutation, Arg1299Cys, in the PLXND1 gene. The mutation affected a highly conserved residue, segregated with the disease in the family and was absent from available SNP databases and ethnic matched controls. in silico comparative modeling revealed that the mutation resides in the N‐terminal segment of the human plexin‐D1 intracellular region which interacts with the catalytic GTPase‐activating protein homology region. The mutation likely destabilizes the intracellular region, perturbing its anchoring and catalytic activity. The phenotype in human PLXND1 mutation is closely related to that of knockout mice for PLXND1, its co‐receptor neuropilin‐1 or its ligand SEMA3C. It is therefore suggested that SEMA3C signaling, propagated through the heterodimer receptor plexin‐D1/neuropilin, is important for truncus arteriosus septation. Confirmation of this observation will require the identification of PLXND1 mutations in additional patients. Exome analysis is valuable for molecular investigation of single patients with congenital heart defects in whom chromosomal copy number variants have been excluded.

Therapy with eculizumab for patients with CD59 p.Cys89Tyr mutation

The objective of this work was to report on the outcome of eculizumab treatment in pediatric patients with recurrent acute predominantly motor, demyelinating neuropathy with conduction block, and chronic hemolysis attributed to p.Cys89Tyr mutation in the CD59 gene.

Congenital valvular defects associated with deleterious mutations in the PLD1 gene

Background The underlying molecular aetiology of congenital heart defects is largely unknown. The aim of this study was to explore the genetic basis of non-syndromic severe congenital valve malformations in two unrelated families. Methods Whole-exome analysis was used to identify the mutations in five patients who suffered from severe valvular malformations involving the pulmonic, tricuspid and mitral valves. The significance of the findings was assessed by studying sporulation of yeast carrying a homologous Phospholipase D (PLD1) mutation, in situ hybridisation in chick embryo and echocardiography and histological examination of hearts of PLD1 knockout mice. Results Three mutations, p.His442Pro, p.Thr495fs32* and c.2882+2T>C, were identified in the PLD1 gene. The mutations affected highly conserved sites in the PLD1 protein and the p.His442Pro mutation produced a strong loss of function phenotype in yeast homologous mutant strain. Here we show that in chick embryos PLD1 expression is confined to the forming heart (E2–E8) and homogeneously expressed all over the heart during days E2–E3. Thereafter its expression decreases, remaining only adjacent to the atrioventricular valves and the right ventricular outflow tract. This pattern of expression follows the known dynamic patterning of apoptosis in the developing heart, consistent with the known role of PLD1 in the promotion of apoptosis. In hearts of PLD1 knockout mice, we detected marked tricuspid regurgitation, right atrial enlargement, and increased flow velocity, narrowing and thickened leaflets of the pulmonic valve. Conclusions The findings support a role for PLD1 in normal heart valvulogenesis.

Hematopoietic stem cell transplantation conditioning with use of rituximab in EBV related lymphoproliferative disorders.

X-linked lymphoproliferative disease (XLP) and IL-2-inducible T cell kinase (ITK) deficiency are rare immunodeficiencies with a spectrum of clinical manifestations. Although there are no official guidelines for allogeneic hematopoietic stem cell transplantation (HSCT) in these patients, previous reports have shown that reduced intensity conditioning regimens provide successful engraftment with limited toxicity. Here, we report on three children with XLP and one with ITK deficiency, who underwent successful HSCT using a rituximab containing conditioning regimen, and review the current literature.

Mutation in the COX4I1 gene is associated with short stature, poor weight gain and increased chromosomal breaks, simulating Fanconi anemia

We describe a novel autosomal recessive form of mitochondrial disease in a child with short stature, poor weight gain, and mild dysmorphic features with highly suspected Fanconi anemia due to a mutation in COX4I1 gene. Whole Exome Sequencing was performed then followed by Sanger confirmation, identified a K101N mutation in COX4I1, segregating with the disease. This nuclear gene encodes the common isoform of cytochrome c oxidase (COX) subunit 4 (COX 4-1), an integral regulatory part of COX (respiratory chain complex IV) the terminal electron acceptor of the mitochondrial respiratory chain. The patients fibroblasts disclosed decreased COX activity, impaired ATP production, elevated ROS production, decreased expression of COX4I1 mRNA and undetectable (COX4) protein. COX activity and ATP production were restored by lentiviral transfection with the wild-type gene. Our results demonstrate the first human mutation in the COX4I1 gene linked to diseases and confirm its role in the pathogenesis. Thus COX4I1 mutations should be considered in any patient with features suggestive of this diagnosis.