Mutations in TUBGCP4 Alter Microtubule Organization via the γ-Tubulin Ring Complex in Autosomal-Recessive Microcephaly with Chorioretinopathy

We have identified TUBGCP4 variants in individuals with autosomal-recessive microcephaly and chorioretinopathy. Whole-exome sequencing performed on one family with two affected siblings and independently on another family with one affected child revealed compound-heterozygous mutations in TUBGCP4. Subsequent Sanger sequencing was performed on a panel of individuals from 12 French families affected by microcephaly and ophthalmic manifestations, and one other individual was identified with compound-heterozygous mutations in TUBGCP4. One synonymous variant was common to all three families and was shown to induce exon skipping; the other mutations were frameshift mutations and a deletion. TUBGCP4 encodes γ-tubulin complex protein 4, a component belonging to the γ-tubulin ring complex (γ-TuRC) and known to regulate the nucleation and organization of microtubules. Functional analysis of individual fibroblasts disclosed reduced levels of the γ-TuRC, altered nucleation and organization of microtubules, abnormal nuclear shape, and aneuploidy. Moreover, zebrafish treated with morpholinos against tubgcp4 were found to have reduced head volume and eye developmental anomalies with chorioretinal dysplasia. In summary, the identification of TUBGCP4 mutations in individuals with microcephaly and a spectrum of anomalies in eye development, particularly photoreceptor anomalies, provides evidence of an important role for the γ-TuRC in brain and eye development.     

Identification of a Ninein (NIN) mutation in a family with spondyloepimetaphyseal dysplasia with joint laxity (leptodactylic type)-like phenotype

Spondyloepimetaphyseal dysplasia with joint laxity-leptodactylic type (SEMDJL2) is an autosomal dominant skeletal dysplasia which is characterized by midface hypoplasia, short stature, joint laxity with dislocations, genua valga, progressive scoliosis, and slender fingers. Recently, heterozygous missense mutations in KIF22, a gene which encodes a member of the kinesin-like protein family, have been identified in sporadic as well as familial cases of SEMDJL2. In the present study homozygosity mapping and whole-exome sequencing were combined to analyze a consanguineous family with a phenotype resembling SEMDJL2. We identified homozygous missense mutations in the two nearby genes NIN (Ninein) and POLE2 (DNA polymerase epsilon subunit B) which segregate with the disease in the family and were not present in 500 healthy control individuals and in the 1094 control individuals contained within the 1000-genomes database. We present several lines of evidence that mutant Ninein is most likely causative for the SEMDJL2-like phenotype. The centrosomal protein NIN shows a functional relationship with KIF22 and other proteins associated with chromosome congression/movement, centrosomal function, and ciliogenesis, which have been associated with skeletal dysplasias. Moreover, compound heterozygous missense mutations at more N-terminal positions of Ninein have very recently been identified in a family with microcephalic primordial dwarfism. Together with the present report this strongly supports a fundamental role of Ninein in skeletal development.     

Mutations in CKAP2L,the Human Homolog of the Mouse Radmis Gene,Cause Filippi Syndrome

Filippi syndrome is a rare, presumably autosomal-recessive disorder characterized by microcephaly, pre- and postnatal growth failure, syndactyly, and distinctive facial features, including a broad nasal bridge and underdeveloped alae nasi. Some affected individuals have intellectual disability, seizures, undescended testicles in males, and teeth and hair abnormalities. We performed homozygosity mapping and whole-exome sequencing in a Sardinian family with two affected children and identified a homozygous frameshift mutation, c.571dupA (p.Ile191Asnfs^∗6), in CKAP2L, encoding the protein cytoskeleton-associated protein 2-like (CKAP2L). The function of this protein was unknown until it was rediscovered in mice as Radmis (radial fiber and mitotic spindle) and shown to play a pivotal role in cell division of neural progenitors. Sanger sequencing of CKAP2L in a further eight unrelated individuals with clinical features consistent with Filippi syndrome revealed biallelic mutations in four subjects. In contrast to wild-type lymphoblastoid cell lines (LCLs), dividing LCLs established from the individuals homozygous for the c.571dupA mutation did not show CKAP2L at the spindle poles. Furthermore, in cells from the affected individuals, we observed an increase in the number of disorganized spindle microtubules owing to multipolar configurations and defects in chromosome segregation. The observed cellular phenotypes are in keeping with data from in vitro and in vivo knockdown studies performed in human cells and mice, respectively. Our findings show that loss-of-function mutations in CKAP2L are a major cause of Filippi syndrome.     

Loss-of-Function Mutations in WDR73 Are Responsible for Microcephaly and Steroid-Resistant Nephrotic Syndrome: Galloway-Mowat Syndrome

Galloway-Mowat syndrome is a rare autosomal-recessive condition characterized by nephrotic syndrome associated with microcephaly and neurological impairment. Through a combination of autozygosity mapping and whole-exome sequencing, we identified WDR73 as a gene in which mutations cause Galloway-Mowat syndrome in two unrelated families. WDR73 encodes a WD40-repeat-containing protein of unknown function. Here, we show that WDR73 was present in the brain and kidney and was located diffusely in the cytoplasm during interphase but relocalized to spindle poles and astral microtubules during mitosis. Fibroblasts from one affected child and WDR73-depleted podocytes displayed abnormal nuclear morphology, low cell viability, and alterations of the microtubule network. These data suggest that WDR73 plays a crucial role in the maintenance of cell architecture and cell survival. Altogether, WDR73 mutations cause Galloway-Mowat syndrome in a particular subset of individuals presenting with late-onset nephrotic syndrome, postnatal microcephaly, severe intellectual disability, and homogenous brain MRI features. WDR73 is another example of a gene involved in a disease affecting both the kidney glomerulus and the CNS.     

Mutations in SPATA5 Are Associated with Microcephaly,Intellectual Disability,Seizures, and Hearing Loss

Using whole-exome sequencing, we have identified in ten families 14 individuals with microcephaly, developmental delay, intellectual disability, hypotonia, spasticity, seizures, sensorineural hearing loss, cortical visual impairment, and rare autosomal-recessive predicted pathogenic variants in spermatogenesis-associated protein 5 (SPATA5). SPATA5 encodes a ubiquitously expressed member of the ATPase associated with diverse activities (AAA) protein family and is involved in mitochondrial morphogenesis during early spermatogenesis. It might also play a role in post-translational modification during cell differentiation in neuronal development. Mutations in SPATA5 might affect brain development and function, resulting in microcephaly, developmental delay, and intellectual disability.     

GJC2 Missense Mutations Cause Human Lymphedema

Lymphedema is the clinical manifestation of defects in lymphatic structure or function. Mutations identified in genes regulating lymphatic development result in inherited lymphedema. No mutations have yet been identified in genes mediating lymphatic function that result in inherited lymphedema. Survey microarray studies comparing lymphatic and blood endothelial cells identified expression of several connexins in lymphatic endothelial cells. Additionally, gap junctions are implicated in maintaining lymphatic flow. By sequencing GJA1, GJA4, and GJC2 in a group of families with dominantly inherited lymphedema, we identified six probands with unique missense mutations in GJC2 (encoding connexin [Cx] 47). Two larger families cosegregate lymphedema and GJC2 mutation (LOD score = 6.5). We hypothesize that missense mutations in GJC2 alter gap junction function and disrupt lymphatic flow. Until now, GJC2 mutations were only thought to cause dysmyelination, with primary expression of Cx47 limited to the central nervous system. The identification of GJC2 mutations as a cause of primary lymphedema raises the possibility of novel gap-junction-modifying agents as potential therapy for some forms of lymphedema.     

Mutations in PYCR2, Encoding Pyrroline-5-Carboxylate Reductase 2, Cause Microcephaly and Hypomyelination

Despite recent advances in understanding the genetic bases of microcephaly, a large number of cases of microcephaly remain unexplained, suggesting that many microcephaly syndromes and associated genes have yet to be identified. Here, we report mutations in PYCR2, which encodes an enzyme in the proline biosynthesis pathway, as the cause of a unique syndrome characterized by postnatal microcephaly, hypomyelination, and reduced cerebral white-matter volume. Linkage mapping and whole-exome sequencing identified homozygous mutations (c.355C>T [p.Arg119Cys] and c.751C>T [p.Arg251Cys]) in PYCR2 in the affected individuals of two consanguineous families. A lymphoblastoid cell line from one affected individual showed a strong reduction in the amount of PYCR2. When mutant cDNAs were transfected into HEK293FT cells, both variant proteins retained normal mitochondrial localization but had lower amounts than the wild-type protein, suggesting that the variant proteins were less stable. A PYCR2-deficient HEK293FT cell line generated by genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that PYCR2 loss of function led to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress. Morpholino-based knockdown of a zebrafish PYCR2 ortholog, pycr1b, recapitulated the human microcephaly phenotype, which was rescued by wild-type human PYCR2 mRNA, but not by mutant mRNAs, further supporting the pathogenicity of the identified variants. Hypomyelination and the absence of lax, wrinkly skin distinguishes this condition from that caused by previously reported mutations in the gene encoding PYCR2’s isozyme, PYCR1, suggesting a unique and indispensable role for PYCR2 in the human CNS during development.     

KIF18B is a cell type–specific regulator of spindle orientation in the epidermis

Proper spindle orientation is required for asymmetric cell division and the establishment of complex tissue architecture. In the developing epidermis, spindle orientation requires a conserved cortical protein complex of LGN/NuMA/dynein-dynactin. However, how microtubule dynamics are regulated to interact with this machinery and properly position the mitotic spindle is not fully understood. Furthermore, our understanding of the processes that link spindle orientation during asymmetric cell division to cell fate specification in distinct tissue contexts remains incomplete. We report a role for the microtubule catastrophe factor KIF18B in regulating microtubule dynamics to promote spindle orientation in keratinocytes. During mitosis, KIF18B accumulates at the cell cortex, colocalizing with the conserved spindle orientation machinery. In vivo we find that KIF18B is required for oriented cell divisions within the hair placode, the first stage of hair follicle morphogenesis, but is not essential in the interfollicular epidermis. Disrupting spindle orientation in the placode, using mutations in either KIF18B or NuMA, results in aberrant cell fate marker expression of hair follicle progenitor cells. These data functionally link spindle orientation to cell fate decisions during hair follicle morphogenesis. Taken together, our data demonstrate a role for regulated microtubule dynamics in spindle orientation in epidermal cells. This work also highlights the importance of spindle orientation during asymmetric cell division to dictate cell fate specification.     

Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies

Applying a next-generation sequencing assay targeting 145 cancer-relevant genes in 40 colorectal cancer and 24 non-small cell lung cancer formalin-fixed paraffin-embedded tissue specimens identified at least one clinically relevant genomic alteration in 59% of the samples and revealed two gene fusions, C2orf44-ALK in a colorectal cancer sample and KIF5B-RET in a lung adenocarcinoma. Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions (2.0%; 95% CI 0.8-3.1%). Cells expressing oncogenic KIF5B-RET are sensitive to multi-kinase inhibitors that inhibit RET.     

Recurrent dominant mutations affecting two adjacent residues in the motor domain of the monomeric kinesin KIF22 result in skeletal dysplasia and joint laxity

Spondyloepimetaphyseal dysplasia with joint laxity, leptodactylic type (lepto-SEMDJL, aka SEMDJL, Hall type), is an autosomal dominant skeletal disorder that, in spite of being relatively common among skeletal dysplasias, has eluded molecular elucidation so far. We used whole-exome sequencing of five unrelated individuals with lepto-SEMDJL to identify mutations in KIF22 as the cause of this skeletal condition. Missense mutations affecting one of two adjacent amino acids in the motor domain of KIF22 were present in 20 familial cases from eight families and in 12 other sporadic cases. The skeletal and connective tissue phenotype produced by these specific mutations point to functions of KIF22 beyond those previously ascribed functions involving chromosome segregation. Although we have found Kif22 to be strongly upregulated at the growth plate, the precise pathogenetic mechanisms remain to be elucidated.     

Wide phenotypic spectrum in axonal Charcot–Marie–Tooth neuropathy type 2 patients with <Emphasis Type="Italic">KIF5A</Emphasis> mutations

The kinesin heavy chain isoform 5A (KIF5A) gene, which encodes a microtubule-based motor protein, plays an important role in the transport of organelles in the nerve cells. Mutations in the KIF5A showed a wide phenotypic spectrum from hereditary spastic paraplegia (HSP) to axonal Charcot–Marie–Tooth peripheral neuropathy type 2 (CMT2). This study identified three pathogenic KIF5A mutations in Korean CMT2 patients by whole exome sequencing. Two mutations (p.Arg204Trp and p.Arg280His) were previously reported, but p.Leu558Pro was determined to be a novel de novo mutation. All the mutations were not observed in the healthy controls and were located in highly conserved domains among vertebrate species. The p.Arg204Trp mutation was identified from a CMT2 patient with additional complex phenotypes of HSP, ataxia, fatigability and pyramidal sign, but the p.Arg280His and p.Leu588Pro mutations were identified in each axonal CMT2 patient. The p.Arg204Trp mutation was previously reported in a HSP patient with no CMT symptom. The p.Arg280His mutation was reported in a CMT2 patient, which was similarly with our case. However, it was also once reported in a HSP patient with pes cavus. As the first report in Korea, this study identified three KIF5A mutations as the underlying cause of axonal peripheral neuropathy with or without the HSP phenotype. We confirmed a wide inter- and intra-allelic phenotypic spectrum by the mutations in the KIF5A.     

Whole-exome sequencing identifies mutations of KIF22 in spondyloepimetaphyseal dysplasia with joint laxity, leptodactylic type

Spondyloepimetaphyseal dysplasia with joint laxity (SEMDJL), leptodactylic (lepto-SEMDJL) or Hall type, is an autosomal-dominant skeletal dysplasia manifesting with short stature, joint laxity with dislocation(s), limb malalignment, and spinal deformity. Its causative gene mutation has not yet been discovered. We captured and sequenced the exomes of eight affected individuals in six unrelated kindreds (three individuals in a family and five simplex individuals). Five novel sequence variants in KIF22, which encodes a member of the kinesin-like protein family, were identified in seven individuals. Sanger sequencing of KIF22 confirmed that c.443C>T (p.Pro148Ser) cosegregated with the phenotype in the affected individuals in the family; c.442C>T (p.Pro148Leu) or c.446G>A (p.Arg149Gln) was present in four of five simplex individuals, but was absent in unaffected individuals in their family and 505 normal cohorts. KIF22 mRNA was detected in human bone, cartilage, joint capsule, ligament, skin, and primary cultured chondrocytes. In silico analysis of KIF22 protein structure indicates that Pro148 and Arg149 are important in maintaining hydrogen bonds in the ATP binding and motor domains of KIF22. We conclude that these mutations in KIF22 cause lepto-SEMDJL.     

Haploinsufficiency of a spliceosomal GTPase encoded by EFTUD2 causes mandibulofacial dysostosis with microcephaly

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare sporadic syndrome comprising craniofacial malformations, microcephaly, developmental delay, and a recognizable dysmorphic appearance. Major sequelae, including choanal atresia, sensorineural hearing loss, and cleft palate, each occur in a significant proportion of affected individuals. We present detailed clinical findings in 12 unrelated individuals with MFDM; these 12 individuals compose the largest reported cohort to date. To define the etiology of MFDM, we employed whole-exome sequencing of four unrelated affected individuals and identified heterozygous mutations or deletions of EFTUD2 in all four. Validation studies of eight additional individuals with MFDM demonstrated causative EFTUD2 mutations in all affected individuals tested. A range of EFTUD2-mutation types, including null alleles and frameshifts, is seen in MFDM, consistent with haploinsufficiency; segregation is de novo in all cases assessed to date. U5-116kD, the protein encoded by EFTUD2, is a highly conserved spliceosomal GTPase with a central regulatory role in catalytic splicing and post-splicing-complex disassembly. MFDM is the first multiple-malformation syndrome attributed to a defect of the major spliceosome. Our findings significantly extend the range of reported spliceosomal phenotypes in humans and pave the way for further investigation in related conditions such as Treacher Collins syndrome.     

Role of the kinesin-2 family protein, KIF3, during mitosis

During mitosis, kinesin and dynein motor proteins play critical roles in the equal segregation of chromosomes between two daughter cells. Kinesin-2 is composed of two microtubule-based motor subunits, KIF3A/3B, and a kinesin-associated protein known as KAP3, which links KIF3A/3B to cargo that is carried to cellular organelles along microtubules in interphase cells. We have shown here that the kinesin-2 complex is localized with components of the mitotic apparatus such as spindle microtubules and centrosomes. Furthermore, we found that expression of a mutant KIF3B, which is able to associate with KIF3A but not KAP3 in NIH3T3 cells, caused chromosomal aneuploidy and abnormal spindle formation. Our data suggested that the kinesin-2 complex plays an important role not only in interphase but also in mitosis.     

Kinesin-6 family motor KIF20A regulates central spindle assembly and acrosome biogenesis in mouse spermatogenesis

Kinesin-6 KIF20A is essential for microtubule organization and central spindle assembly during cytokinesis. However, the functions of KIF20A in meiotic division and spermatogenesis remain elusive. Here, we report that kinesin-6 KIF20A locates at the microtubules in mouse spermatogenic cells and co-localizes with the spindle midzone and midbody. We demonstrate that central spindle organization and chromosomal stability are regulated by KIF20A in male meiotic division. KIF20A inhibition leads to the defects in central spindle assembly and cytokinetic abscission, and finally results in the increase of aneuploid cells and the alteration of cell populations in the spermatogenic cells. Furthermore, we have revealed that kinesin-6 KIF20A is associated with the formation and maturation of the acrosomes during spermatogenesis. Our findings have identified the specific roles of KIF20A in central spindle organization in meiotic division.     

Mutation analysis in Rett syndrome

Rett syndrome is an X-linked dominant neurodevelopmental disorder caused by mutations in the MECP2 gene. Mutations have been demonstrated in more than 80% of females with typical features of Rett syndrome. We identified mutations in the MECP2 gene and documented the clinical manifestations in 65 Rett syndrome patients to characterize the genotype-phenotype spectrum. Bidirectional sequencing of the entire MECP2 coding region was performed. We diagnosed 65 patients with MECP2 mutations. Of these, 15 mutations had been reported previously and 13 are novel. Two patients have multiple deletions within the MECP2 gene. Eight common mutations were found in 43 of 65 patients (66.15%). The majority of patients with identified mutations have the classic Rett phenotype, and several had atypical phenotypes. MECP2 analysis identified mutations in almost all cases of typical Rett syndrome, as well as in some with atypical phenotypes. Eleven (20.4%) of the 54 patients with defined mutations and in whom phenotypic data were obtained did not develop acquired microcephaly. Hence, microcephaly at birth or absence of acquired microcephaly does not obviate the need for MECP2 analysis. We have initiated cascade testing starting with PCR analysis for common mutations followed by sequencing, when necessary. Analysis of common mutations before sequencing the entire gene is anticipated to be the most efficacious strategy to identify Rett syndrome gene mutations.     

KIF1A, an axonal transporter of synaptic vesicles, is mutated in hereditary sensory and autonomic neuropathy type 2

Hereditary sensory and autonomic neuropathy type II (HSANII) is a rare autosomal-recessive disorder characterized by peripheral nerve degeneration resulting in a severe distal sensory loss. Although mutations in FAM134B and the HSN2 exon of WNK1 were associated with HSANII, the etiology of a substantial number of cases remains unexplained. In addition, the functions of WNK1/HSN2 and FAM134B and their role in the peripheral nervous system remain poorly understood. Using a yeast two-hybrid screen, we found that KIF1A, an axonal transporter of synaptic vesicles, interacts with the domain encoded by the HSN2 exon. In parallel to this screen, we performed genome-wide homozygosity mapping in a consanguineous Afghan family affected by HSANII and identified a unique region of homozygosity located on chromosome 2q37.3 and spanning the KIF1A gene locus. Sequencing of KIF1A in this family revealed a truncating mutation segregating with the disease phenotype. Subsequent sequencing of KIF1A in a series of 112 unrelated patients with features belonging to the clinical spectrum of ulcero-mutilating sensory neuropathies revealed truncating mutations in three additional families, thus indicating that mutations in KIF1A are a rare cause of HSANII. Similarly to WNK1 mutations, pathogenic mutations in KIF1A were almost exclusively restricted to an alternatively spliced exon. This study provides additional insights into the molecular pathogenesis of HSANII and highlights the potential biological relevance of alternative splicing in the peripheral sensory nervous system.     

Protein-truncating mutations in ASPM cause variable reduction in brain size

Mutations in the ASPM gene at the MCPH5 locus are expected to be the most common cause of human autosomal recessive primary microcephaly (MCPH), a condition in which there is a failure of normal fetal brain development, resulting in congenital microcephaly and mental retardation. We have performed the first comprehensive mutation screen of the 10.4-kb ASPM gene, identifying all 19 mutations in a cohort of 23 consanguineous families. Mutations occurred throughout the ASPM gene and were all predicted to be protein truncating. Phenotypic variation in the 51 affected individuals occurred in the degree of microcephaly (5-11 SDs below normal) and of mental retardation (mild to severe) but appeared independent of mutation position.     

Next generation sequencing reveals novel homozygous frameshift in PUS7 and splice acceptor variants in AASS gene leading to intellectual disability,developmental delay,dysmorphic feature and microcephaly

Intellectual developmental disorder with abnormal behavior, microcephaly and short stature (IDDABS), (OMIM# 618342) is an autosomal recessive condition described as developmental delay, poor or absent speech, intellectual disability, short stature, mild to progressive microcephaly, delayed psychomotor development, hyperactivity, seizure, along with mild to swear aggressive behavior. Homozygous frameshift mutation in Pseudouridine Synthase 7, Putative; (PUS7) OMIM# 616,261 NM_019042.3 and splice acceptor variants in Alpha-Aminoadipic Semialdehyde Synthase; (AASS) OMIM# 605,113 NM_005763.3 was funded. Whole exome sequencing (WES) technique was used as tool to identify the molecular diagnostic test. Different bioinformatics analysis done for WES data and we identified two novel mutations one as frameshift mutation c.606_607delGA, p.Ser282CysfsTer9 in the PUS7 gene and splice acceptor variants c.1767–1 G > A in the AASS gene has been reported. The pattern of family segregation maintained the pathogenicity of this variation associated with abnormal behavior, intellectual developmental disorder, microcephaly along with short stature IDDABS. Further, the WES data was validated in the family having other affected individuals and healthy controls (n = 100) was done using Sanger sequencing. Finally, our results further explained the role of WES in the disease diagnosis and elucidated that the mutation in PUS7 and AASS genes may lead an important role for the development of IDDABS in Saudi family.