ßIV Spectrinopathies Cause Profound Intellectual Disability, Congenital Hypotonia, and Motor Axonal Neuropathy.

ßIV spectrin links ankyrinG (AnkG) and clustered ion channels at axon initial segments (AISs) and nodes of Ranvier to the axonal cytoskeleton. Here, we report bi-allelic pathogenic SPTBN4 variants (three homozygous and two compound heterozygous) that cause a severe neurological syndrome that includes congenital hypotonia, intellectual disability, and motor axonal and auditory neuropathy. We introduced these variants into ßIV spectrin, expressed these in neurons, and found that 5/7 were loss-of-function variants disrupting AIS localization or abolishing phosphoinositide binding. Nerve biopsies from an individual with a loss-of-function variant had reduced nodal Na+ channels and no nodal KCNQ2 K+ channels. Modeling the disease in mice revealed that although ankyrinR (AnkR) and ßI spectrin can cluster Na+ channels and partially compensate for the loss of AnkG and ßIV spectrin at nodes of Ranvier, AnkR and ßI spectrin cannot cluster KCNQ2- and KCNQ3-subunit-containing K+ channels. Our findings define a class of spectrinopathies and reveal the molecular pathologies causing nervous-system dysfunction.

ECHS1 disease in two unrelated families of Samoan descent: Common variant - rare disorder.

Mutations in the short-chain enoyl-CoA hydratase (SCEH) gene, ECHS1, cause a rare autosomal recessive disorder of valine catabolism. Patients usually present with developmental delay, regression, dystonia, feeding difficulties, and abnormal MRI with bilateral basal ganglia involvement. We present clinical, biochemical, molecular, and functional data for four affected patients from two unrelated families of Samoan descent with identical novel compound heterozygous mutations. Family 1 has three affected boys while Family 2 has an affected daughter, all with clinical and MRI findings of Leigh syndrome and intermittent episodes of acidosis and ketosis. WES identified a single heterozygous variant in ECHS1 at position c.832G > A (p.Ala278Thr). However, western blot revealed significantly reduced ECHS1 protein for all affected family members. Decreased SCEH activity in fibroblasts and a mild increase in marker metabolites in urine further supported ECHS1 as the underlying gene defect. Additional investigations at the DNA (aCGH, WGS) and RNA (qPCR, RT-PCR, RNA-Seq, RNA-Array) level identified a silent, common variant at position c.489G > A (p.Pro163=) as the second mutation. This substitution, present at high frequency in the Samoan population, is associated with decreased levels of normally spliced mRNA. To our understanding, this is the first report of a novel, hypomorphic allele c.489G > A (p.Pro163=), associated with SCEH deficiency.

Rubinstein-Taybi syndrome in diverse populations.

Rubinstein-Taybi syndrome (RSTS) is an autosomal dominant disorder, caused by loss-of-function variants in CREBBP or EP300. Affected individuals present with distinctive craniofacial features, broad thumbs and/or halluces, and intellectual disability. RSTS phenotype has been well characterized in individuals of European descent but not in other populations. In this study, individuals from diverse populations with RSTS were assessed by clinical examination and facial analysis technology. Clinical data of 38 individuals from 14 different countries were analyzed. The median age was 7 years (age range: 7 months to 47 years), and 63% were females. The most common phenotypic features in all population groups included broad thumbs and/or halluces in 97%, convex nasal ridge in 94%, and arched eyebrows in 92%. Face images of 87 individuals with RSTS (age range: 2 months to 47 years) were collected for evaluation using facial analysis technology. We compared images from 82 individuals with RSTS against 82 age- and sex-matched controls and obtained an area under the receiver operating characteristic curve (AUC) of 0.99 (p < .001), demonstrating excellent discrimination efficacy. The discrimination was, however, poor in the African group (AUC: 0.79; p = .145). Individuals with EP300 variants were more effectively discriminated (AUC: 0.95) compared with those with CREBBP variants (AUC: 0.93). This study shows that clinical examination combined with facial analysis technology may enable earlier and improved diagnosis of RSTS in diverse populations.

Lack of functional P-selectin ligand exacerbates Salmonella serovar typhimurium infection.

The selectin family of adhesion molecules mediates the recruitment of immune cells to the site of inflammation, which is critical for host survival of infection. To characterize the role of selectins in host defense against Salmonella Typhimurium infection, wild-type (WT) mice and mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), P-, E-, or L-selectin, or the glycosyltransferase C2GlcNAcT-I (core 2) were infected using a Salmonella acute gastroenteritis model. Mice were monitored for survival and assessed for intestinal inflammation at 1 and 4 days postinfection. Infected mice lacking core 2, PSGL-1, or P-selectin showed a more pronounced morbidity and a significantly higher mortality rate associated with higher bacterial load and proinflammatory cytokine production, including that of TNF-alpha, MCP-1, and IL-6, from the colons at 4 days postinfection as compared with WT control. Surprisingly, at 1 day postinfection, more severe inflammation and higher neutrophil infiltration were observed in the ceca of mice lacking core 2, PSGL-1, or P-selectin compared with WT control. Enhanced levels of alpha(4)beta(7)(+) and MAdCAM-1(+) cells were observed in the ceca of infected mice lacking core 2, PSGL-1, or P-selectin. Neutrophil recruitment, cecal inflammation, and mortality rates were dramatically reduced in infected P-selectin knockout mice receiving blocking mAb to alpha(4)beta(7) integrin, indicating that this alternative adhesion molecule may attempt to compensate for the loss of selectins in neutrophil recruitment. These results demonstrate a definitive phenotypic abnormality in mice lacking core 2, PSGL-1, or P-selectin, suggesting that the interaction of functional PSGL-1 with P-selectin is an important process in host defense against Salmonella infection.

Characteristics of Orofacial Clefting in Hawai'i.

Orofacial clefts are birth defects that require a multi-disciplinary approach for repair and ongoing management as there are often concomitant chronic health issues. Orofacial clefts can occur as an isolated finding, in combination with other anomalies, or as part of a genetic syndrome. When occurring as part of a genetic syndrome, the complexity of management increases and has lifelong implications for these individuals, their families, and their health care providers. Understanding factors related to the occurrence of syndromic orofacial clefting is important for birth defect research and for health care needs assessment and planning. Many research groups have addressed these issues by studying different populations and focusing on different questions. This study was a retrospective chart review of children with orofacial clefts cared for at a pediatric tertiary care center in Hawai'i to evaluate the proportion of isolated and syndromic clefts in the unique population of Hawai'i. The prevalence of syndromic and isolated clefts were then correlated with ethnicity and compared to the prevalence in other studies. Our goal was to increase knowledge about orofacial clefting in the population of Hawai'i. The proportion of isolated orofacial clefting in a population of patients with orofacial clefting cared for at a craniofacial clinic is similar to birth defect registry data for the Hawaiian Islands (59% vs 58%). Pacific Islanders in our study and prior study have a lower proportion of isolated clefts, suggesting that there are more craniofacial patients with syndromic and complex needs in this population. Further study is needed to clarify the etiologic factors.

Loss-of-function mutations in Lysyl-tRNA synthetase cause various leukoencephalopathy phenotypes.

OBJECTIVE: To expand the clinical spectrum of lysyl-tRNA synthetase (KARS) gene-related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment. METHODS: Whole-exome sequencing was performed on index patients from 4 unrelated families with leukoencephalopathy. Candidate pathogenic variants and their cosegregation were confirmed by Sanger sequencing. Effects of mutations on KARS protein function were examined by aminoacylation assays and yeast complementation assays. RESULTS: Common clinical features of the patients in this study included impaired cognitive ability, seizure, hypotonia, ataxia, and abnormal brain imaging, suggesting that the CNS involvement is the main clinical presentation. Six previously unreported and 1 known KARS mutations were identified and cosegregated in these families. Two patients are compound heterozygous for missense mutations, 1 patient is homozygous for a missense mutation, and 1 patient harbored an insertion mutation and a missense mutation. Functional and structural analyses revealed that these mutations impair aminoacylation activity of lysyl-tRNA synthetase, indicating that defective KARS function is responsible for the phenotypes in these individuals. CONCLUSIONS: Our results demonstrate that patients with loss-of-function KARS mutations can manifest CNS disorders, thus broadening the phenotypic spectrum associated with KARS-related disease.

Bilateral radial ulnar synostosis and vertebral anomalies in a child with a de novo 16p13.3 interstitial deletion.

We describe an 8-year-old boy with developmental delay, clinical bilateral radial ulnar synostosis, Klippel-Feil anomaly, and other vertebral deformities who was found to have a de novo deletion of 114.5kb at 16p13.3. The deletion contains five genes and three miRNAs. The genes are E4F1, DNASE1L2, ECI1, RNPS1, and ABCA3; miRNAs are MIR3677, MIR940, and MIR4717. The specific deletion has never been previously reported. We describe the phenotype of the boy and review the genes in the deleted region. None of the regulatory elements have any known linkage to skeletal formation and/or maintenance. We believe this deletion is causative given that it was de novo and that this patient cannot be easily explained as having any other specific recognizable pattern of human malformation.

A novel mutation in the HSD17B10 gene of a 10-year-old boy with refractory epilepsy, choreoathetosis and learning disability.

Hydroxysteroid (17beta) dehydrogenase 10 (HSD10) is a mitochondrial multifunctional enzyme encoded by the HSD17B10 gene. Missense mutations in this gene result in HSD10 deficiency, whereas a silent mutation results in mental retardation, X-linked, syndromic 10 (MRXS10). Here we report a novel missense mutation found in the HSD17B10 gene, namely c.194T>C transition (rs104886492), brought about by the loss of two forked methyl groups of valine 65 in the HSD10 active site. The affected boy, who possesses mutant HSD10 (p.V65A), has a neurological syndrome with metabolic derangements, choreoathetosis, refractory epilepsy and learning disability. He has no history of acute decompensation or metabolic acidosis whereas his urine organic acid profile, showing elevated levels of 2-methyl-3-hydroxybutyrate and tiglylglycine, is characteristic of HSD10 deficiency. His HSD10 activity was much lower than the normal control level, with normal ß-ketothiolase activity. The c.194T>C mutation in HSD17B10 can be identified by the restriction fragment polymorphism analysis, thereby facilitating the screening of this novel mutation in individuals with intellectual disability of unknown etiology and their family members much easier. The patient's mother is an asymptomatic carrier, and has a mixed ancestry (Hawaiian, Japanese and Chinese). This demonstrates that HSD10 deficiency patients are not confined to a particular ethnicity although previously reported cases were either Spanish or German descendants.

Inverted genomic segments and complex triplication rearrangements are mediated by inverted repeats in the human genome.

We identified complex genomic rearrangements consisting of intermixed duplications and triplications of genomic segments at the MECP2 and PLP1 loci. These complex rearrangements were characterized by a triplicated segment embedded within a duplication in 11 unrelated subjects. Notably, only two breakpoint junctions were generated during each rearrangement formation. All the complex rearrangement products share a common genomic organization, duplication-inverted triplication-duplication (DUP-TRP/INV-DUP), in which the triplicated segment is inverted and located between directly oriented duplicated genomic segments. We provide evidence that the DUP-TRP/INV-DUP structures are mediated by inverted repeats that can be separated by >300 kb, a genomic architecture that apparently leads to susceptibility to such complex rearrangements. A similar inverted repeat-mediated mechanism may underlie structural variation in many other regions of the human genome. We propose a mechanism that involves both homology-driven events, via inverted repeats, and microhomologous or nonhomologous events.

Regulation of type III secretion hierarchy of translocators and effectors in attaching and effacing bacterial pathogens.

Human enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and the mouse pathogen Citrobacter rodentium (CR) belong to the family of attaching and effacing (A/E) bacterial pathogens. They possess the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system. These pathogens secrete a number of proteins into culture media, including type III effector proteins and translocators that are required for the translocation of effectors into host cells. Preliminary evidence indicated that the LEE-encoded SepL and Rorf6/SepD may form a molecular switch that controls the secretion of translocators and effectors in CR. Here, we show that SepL and SepD indeed perform this function in A/E pathogens such as EHEC and EPEC. Their sepL and sepD mutants do not secrete translocators but exhibit enhanced secretion of effectors. We demonstrate that SepL and SepD interact with each other and that both SepL and SepD are localized to the bacterial membranes. Furthermore, we demonstrate that culture media influence the type III secretion profile of EHEC, EPEC, and CR and that low-calcium concentrations inhibit secretion of translocators but promote the secretion of effectors, similar to effects on type III secretion by mutations in sepL and sepD. However, the secretion profile of the sepD and sepL mutants is not affected by these culture conditions. Collectively, our results suggest that SepL and SepD not only are necessary for efficient translocator secretion in A/E pathogens but also control a switch from translocator to effector secretion by sensing certain environmental signals such as low calcium.

Dissecting virulence: systematic and functional analyses of a pathogenicity island.

Bacterial pathogenicity islands (PAI) often encode both effector molecules responsible for disease and secretion systems that deliver these effectors to host cells. Human enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and the mouse pathogen Citrobacter rodentium (CR) possess the locus of enterocyte effacement (LEE) PAI. We systematically mutagenized all 41 CR LEE genes and functionally characterized these mutants in vitro and in a murine infection model. We identified 33 virulence factors, including two virulence regulators and a hierarchical switch for type III secretion. In addition, 7 potential type III effectors encoded outside the LEE were identified by using a proteomics approach. These non-LEE effectors are encoded by three uncharacterized PAIs in EHEC O157, suggesting that these PAIs act cooperatively with the LEE in pathogenesis. Our findings provide significant insights into bacterial virulence mechanisms and disease.