Biallelic DAW1 variants cause a motile ciliopathy characterized by laterality defects and subtle ciliary beating abnormalities.

PURPOSE: The clinical spectrum of motile ciliopathies includes laterality defects, hydrocephalus, and infertility as well as primary ciliary dyskinesia when impaired mucociliary clearance results in otosinopulmonary disease. Importantly, approximately 30% of patients with primary ciliary dyskinesia lack a genetic diagnosis. METHODS: Clinical, genomic, biochemical, and functional studies were performed alongside in vivo modeling of DAW1 variants. RESULTS: In this study, we identified biallelic DAW1 variants associated with laterality defects and respiratory symptoms compatible with motile cilia dysfunction. In early mouse embryos, we showed that Daw1 expression is limited to distal, motile ciliated cells of the node, consistent with a role in left-right patterning. daw1 mutant zebrafish exhibited reduced cilia motility and left-right patterning defects, including cardiac looping abnormalities. Importantly, these defects were rescued by wild-type, but not mutant daw1, gene expression. In addition, pathogenic DAW1 missense variants displayed reduced protein stability, whereas DAW1 loss-of-function was associated with distal type 2 outer dynein arm assembly defects involving axonemal respiratory cilia proteins, explaining the reduced cilia-induced fluid flow in particle tracking velocimetry experiments. CONCLUSION: Our data define biallelic DAW1 variants as a cause of human motile ciliopathy and determine that the disease mechanism involves motile cilia dysfunction, explaining the ciliary beating defects observed in affected individuals.

Evidence that the Ser192Tyr/Arg402Gln in cis Tyrosinase gene haplotype is a disease-causing allele in oculocutaneous albinism type 1B (OCA1B).

Oculocutaneous albinism type 1 (OCA1) is caused by pathogenic variants in the TYR (tyrosinase) gene which encodes the critical and rate-limiting enzyme in melanin synthesis. It is the most common OCA subtype found in Caucasians, accounting for ~50% of cases worldwide. The apparent 'missing heritability' in OCA is well described, with ~25-30% of clinically diagnosed individuals lacking two clearly pathogenic variants. Here we undertook empowered genetic studies in an extensive multigenerational Amish family, alongside a review of previously published literature, a retrospective analysis of in-house datasets, and tyrosinase activity studies. Together this provides irrefutable evidence of the pathogenicity of two common TYR variants, p.(Ser192Tyr) and p.(Arg402Gln) when inherited in cis alongside a pathogenic TYR variant in trans. We also show that homozygosity for the p.(Ser192Tyr)/p.(Arg402Gln) TYR haplotype results in a very mild, but fully penetrant, albinism phenotype. Together these data underscore the importance of including the TYR p.(Ser192Tyr)/p.(Arg402Gln) in cis haplotype as a pathogenic allele causative of OCA, which would likely increase molecular diagnoses in this missing heritability albinism cohort by 25-50%.

A biallelic SNIP1 Amish founder variant causes a recognizable neurodevelopmental disorder.

SNIP1 (Smad nuclear interacting protein 1) is a widely expressed transcriptional suppressor of the TGF-ß signal-transduction pathway which plays a key role in human spliceosome function. Here, we describe extensive genetic studies and clinical findings of a complex inherited neurodevelopmental disorder in 35 individuals associated with a SNIP1 NM_024700.4:c.1097A>G, p.(Glu366Gly) variant, present at high frequency in the Amish community. The cardinal clinical features of the condition include hypotonia, global developmental delay, intellectual disability, seizures, and a characteristic craniofacial appearance. Our gene transcript studies in affected individuals define altered gene expression profiles of a number of molecules with well-defined neurodevelopmental and neuropathological roles, potentially explaining clinical outcomes. Together these data confirm this SNIP1 gene variant as a cause of an autosomal recessive complex neurodevelopmental disorder and provide important insight into the molecular roles of SNIP1, which likely explain the cardinal clinical outcomes in affected individuals, defining potential therapeutic avenues for future research.

Biallelic PI4KA variants cause neurological, intestinal and immunological disease.

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα/PI4KA/OMIM:600286) is a lipid kinase generating phosphatidylinositol 4-phosphate (PI4P), a membrane phospholipid with critical roles in the physiology of multiple cell types. PI4KIIIα's role in PI4P generation requires its assembly into a heterotetrameric complex with EFR3, TTC7 and FAM126. Sequence alterations in two of these molecular partners, TTC7 (encoded by TTC7A or TCC7B) and FAM126, have been associated with a heterogeneous group of either neurological (FAM126A) or intestinal and immunological (TTC7A) conditions. Here we show that biallelic PI4KA sequence alterations in humans are associated with neurological disease, in particular hypomyelinating leukodystrophy. In addition, affected individuals may present with inflammatory bowel disease, multiple intestinal atresia and combined immunodeficiency. Our cellular, biochemical and structural modelling studies indicate that PI4KA-associated phenotypical outcomes probably stem from impairment of PI4KIIIα-TTC7-FAM126's organ-specific functions, due to defective catalytic activity or altered intra-complex functional interactions. Together, these data define PI4KA gene alteration as a cause of a variable phenotypical spectrum and provide fundamental new insight into the combinatorial biology of the PI4KIIIα-FAM126-TTC7-EFR3 molecular complex.

Dominant mitochondrial membrane protein-associated neurodegeneration (MPAN) variants cluster within a specific C19orf12 isoform.

Mitochondria membrane protein-associated neurodegeneration (MPAN) neurodegenerative disorder is typically associated with biallelic C19orf12 variants. Here we describe a new and review candidate previous monoallelic de novo C19orf12 variants to define loss of function mutations located in the putative non-membrane spanning C19orf12 isoform as the potential basis of monoallelic MPAN.

No association between SCN9A and monogenic human epilepsy disorders.

Many studies have demonstrated the clinical utility and importance of epilepsy gene panel testing to confirm the specific aetiology of disease, enable appropriate therapeutic interventions, and inform accurate family counselling. Previously, SCN9A gene variants, in particular a c.1921A>T p.(Asn641Tyr) substitution, have been identified as a likely autosomal dominant cause of febrile seizures/febrile seizures plus and other monogenic seizure phenotypes indistinguishable from those associated with SCN1A, leading to inclusion of SCN9A on epilepsy gene testing panels. Here we present serendipitous findings of genetic studies that identify the SCN9A c.1921A>T p.(Asn641Tyr) variant at high frequency in the Amish community in the absence of such seizure phenotypes. Together with findings in UK Biobank these data refute an association of SCN9A with epilepsy, which has important clinical diagnostic implications.

MNS1 variant associated with situs inversus and male infertility.

Ciliopathy disorders due to abnormalities of motile cilia encompass a range of autosomal recessive conditions typified by chronic otosinopulmonary disease, infertility, situs abnormalities and hydrocephalus. Using a combination of genome-wide SNP mapping and whole exome sequencing (WES), we investigated the genetic cause of a form of situs inversus (SI) and male infertility present in multiple individuals in an extended Amish family, assuming that an autosomal recessive founder variant was responsible. This identified a single shared (2.34 Mb) region of autozygosity on chromosome 15q21.3 as the likely disease locus, in which we identified a single candidate biallelic frameshift variant in MNS1 [NM_018365.2: c.407_410del; p.(Glu136Glyfs*16)]. Genotyping of multiple family members identified randomisation of the laterality defects in other homozygous individuals, with all wild type or MNS1 c.407_410del heterozygous carriers being unaffected, consistent with an autosomal recessive mode of inheritance. This study identifies an MNS1 variant as a cause of laterality defects and male infertility in humans, mirroring findings in Mns1-deficient mice which also display male infertility and randomisation of left-right asymmetry of internal organs, confirming a crucial role for MNS1 in nodal cilia and sperm flagella formation and function.

Growth disrupting mutations in epigenetic regulatory molecules are associated with abnormalities of epigenetic aging.

Germline mutations in fundamental epigenetic regulatory molecules including DNA methyltransferase 3 alpha (DNMT3A) are commonly associated with growth disorders, whereas somatic mutations are often associated with malignancy. We profiled genome-wide DNA methylation patterns in DNMT3A c.2312G > A; p.(Arg771Gln) carriers in a large Amish sibship with Tatton-Brown-Rahman syndrome (TBRS), their mosaic father, and 15 TBRS patients with distinct pathogenic de novo DNMT3A variants. This defined widespread DNA hypomethylation at specific genomic sites enriched at locations annotated as genes involved in morphogenesis, development, differentiation, and malignancy predisposition pathways. TBRS patients also displayed highly accelerated DNA methylation aging. These findings were most marked in a carrier of the AML-associated driver mutation p.Arg882Cys. Our studies additionally defined phenotype-related accelerated and decelerated epigenetic aging in two histone methyltransferase disorders: NSD1 Sotos syndrome overgrowth disorder and KMT2D Kabuki syndrome growth impairment. Together, our findings provide fundamental new insights into aberrant epigenetic mechanisms, the role of epigenetic machinery maintenance, and determinants of biological aging in these growth disorders.

Mutations in HYAL2, Encoding Hyaluronidase 2, Cause a Syndrome of Orofacial Clefting and Cor Triatriatum Sinister in Humans and Mice.

Orofacial clefting is amongst the most common of birth defects, with both genetic and environmental components. Although numerous studies have been undertaken to investigate the complexities of the genetic etiology of this heterogeneous condition, this factor remains incompletely understood. Here, we describe mutations in the HYAL2 gene as a cause of syndromic orofacial clefting. HYAL2, encoding hyaluronidase 2, degrades extracellular hyaluronan, a critical component of the developing heart and palatal shelf matrix. Transfection assays demonstrated that the gene mutations destabilize the molecule, dramatically reducing HYAL2 protein levels. Consistent with the clinical presentation in affected individuals, investigations of Hyal2-/- mice revealed craniofacial abnormalities, including submucosal cleft palate. In addition, cor triatriatum sinister and hearing loss, identified in a proportion of Hyal2-/- mice, were also found as incompletely penetrant features in affected humans. Taken together our findings identify a new genetic cause of orofacial clefting in humans and mice, and define the first molecular cause of human cor triatriatum sinister, illustrating the fundamental importance of HYAL2 and hyaluronan turnover for normal human and mouse development.

Recessive nephrocerebellar syndrome on the Galloway-Mowat syndrome spectrum is caused by homozygous protein-truncating mutations of WDR73.

We describe a novel nephrocerebellar syndrome on the Galloway-Mowat syndrome spectrum among 30 children (ages 1.0 to 28 years) from diverse Amish demes. Children with nephrocerebellar syndrome had progressive microcephaly, visual impairment, stagnant psychomotor development, abnormal extrapyramidal movements and nephrosis. Fourteen died between ages 2.7 and 28 years, typically from renal failure. Post-mortem studies revealed (i) micrencephaly without polymicrogyria or heterotopia; (ii) atrophic cerebellar hemispheres with stunted folia, profound granule cell depletion, Bergmann gliosis, and signs of Purkinje cell deafferentation; (iii) selective striatal cholinergic interneuron loss; and (iv) optic atrophy with delamination of the lateral geniculate nuclei. Renal tissue showed focal and segmental glomerulosclerosis and extensive effacement and microvillus transformation of podocyte foot processes. Nephrocerebellar syndrome mapped to 700 kb on chromosome 15, which contained a single novel homozygous frameshift variant (WDR73 c.888delT; p.Phe296Leufs*26). WDR73 protein is expressed in human cerebral cortex, hippocampus, and cultured embryonic kidney cells. It is concentrated at mitotic microtubules and interacts with α-, ß-, and γ-tubulin, heat shock proteins 70 and 90 (HSP-70; HSP-90), and the carbamoyl phosphate synthetase 2/aspartate transcarbamylase/dihydroorotase multi-enzyme complex. Recombinant WDR73 p.Phe296Leufs*26 and p.Arg256Profs*18 proteins are truncated, unstable, and show increased interaction with α- and ß-tubulin and HSP-70/HSP-90. Fibroblasts from patients homozygous for WDR73 p.Phe296Leufs*26 proliferate poorly in primary culture and senesce early. Our data suggest that in humans, WDR73 interacts with mitotic microtubules to regulate cell cycle progression, proliferation and survival in brain and kidney. We extend the Galloway-Mowat syndrome spectrum with the first description of diencephalic and striatal neuropathology.

A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression.

The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.

Hypomorphic PCNA mutation underlies a human DNA repair disorder.

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA's interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration.

Mutations in KPTN cause macrocephaly, neurodevelopmental delay, and seizures.

The proper development of neuronal circuits during neuromorphogenesis and neuronal-network formation is critically dependent on a coordinated and intricate series of molecular and cellular cues and responses. Although the cortical actin cytoskeleton is known to play a key role in neuromorphogenesis, relatively little is known about the specific molecules important for this process. Using linkage analysis and whole-exome sequencing on samples from families from the Amish community of Ohio, we have demonstrated that mutations in KPTN, encoding kaptin, cause a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. Our immunofluorescence analyses in primary neuronal cell cultures showed that endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures and that this association is lost upon introduction of the identified mutations. Taken together, our studies have identified kaptin alterations responsible for macrocephaly and neurodevelopmental delay and define kaptin as a molecule crucial for normal human neuromorphogenesis.

Mutations in B4GALNT1 (GM2 synthase) underlie a new disorder of ganglioside biosynthesis.

Glycosphingolipids are ubiquitous constituents of eukaryotic plasma membranes, and their sialylated derivatives, gangliosides, are the major class of glycoconjugates expressed by neurons. Deficiencies in their catabolic pathways give rise to a large and well-studied group of inherited disorders, the lysosomal storage diseases. Although many glycosphingolipid catabolic defects have been defined, only one proven inherited disease arising from a defect in ganglioside biosynthesis is known. This disease, because of defects in the first step of ganglioside biosynthesis (GM3 synthase), results in a severe epileptic disorder found at high frequency amongst the Old Order Amish. Here we investigated an unusual neurodegenerative phenotype, most commonly classified as a complex form of hereditary spastic paraplegia, present in families from Kuwait, Italy and the Old Order Amish. Our genetic studies identified mutations in B4GALNT1 (GM2 synthase), encoding the enzyme that catalyzes the second step in complex ganglioside biosynthesis, as the cause of this neurodegenerative phenotype. Biochemical profiling of glycosphingolipid biosynthesis confirmed a lack of GM2 in affected subjects in association with a predictable increase in levels of its precursor, GM3, a finding that will greatly facilitate diagnosis of this condition. With the description of two neurological human diseases involving defects in two sequentially acting enzymes in ganglioside biosynthesis, there is the real possibility that a previously unidentified family of ganglioside deficiency diseases exist. The study of patients and animal models of these disorders will pave the way for a greater understanding of the role gangliosides play in neuronal structure and function and provide insights into the development of effective treatment therapies.

SLITRK6 mutations cause myopia and deafness in humans and mice.

Myopia is by far the most common human eye disorder that is known to have a clear, albeit poorly defined, heritable component. In this study, we describe an autosomal-recessive syndrome characterized by high myopia and sensorineural deafness. Our molecular investigation in 3 families led to the identification of 3 homozygous nonsense mutations (p.R181X, p.S297X, and p.Q414X) in SLIT and NTRK-like family, member 6 (SLITRK6), a leucine-rich repeat domain transmembrane protein. All 3 mutant SLITRK6 proteins displayed defective cell surface localization. High-resolution MRI of WT and Slitrk6-deficient mouse eyes revealed axial length increase in the mutant (the endophenotype of myopia). Additionally, mutant mice exhibited auditory function deficits that mirrored the human phenotype. Histological investigation of WT and Slitrk6-deficient mouse retinas in postnatal development indicated a delay in synaptogenesis in Slitrk6-deficient animals. Taken together, our results showed that SLITRK6 plays a crucial role in the development of normal hearing as well as vision in humans and in mice and that its disruption leads to a syndrome characterized by severe myopia and deafness.

Mutation of HERC2 causes developmental delay with Angelman-like features.

BACKGROUND: Deregulation of the activity of the ubiquitin ligase E6AP (UBE3A) is well recognised to contribute to the development of Angelman syndrome (AS). The ubiquitin ligase HERC2, encoded by the HERC2 gene is thought to be a key regulator of E6AP. METHODS AND RESULTS: Using a combination of autozygosity mapping and linkage analysis, we studied an autosomal-recessive neurodevelopmental disorder with some phenotypic similarities to AS, found among the Old Order Amish. Our molecular investigation identified a mutation in HERC2 associated with the disease phenotype. We establish that the encoded mutant HERC2 protein has a reduced half-life compared with its wild-type counterpart, which is associated with a significant reduction in HERC2 levels in affected individuals. CONCLUSIONS: Our data implicate a model in which disruption of HERC2 function relates to a reduction in E6AP activity resulting in neurodevelopmental delay, suggesting a previously unrecognised role of HERC2 in the pathogenesis of AS.

Defective mitochondrial mRNA maturation is associated with spastic ataxia.

In human mitochondria, polyadenylation of mRNA, undertaken by the nuclear-encoded mitochondrial poly(A) RNA polymerase, is essential for maintaining mitochondrial gene expression. Our molecular investigation of an autosomal-recessive spastic ataxia with optic atrophy, present among the Old Order Amish, identified a mutation of MTPAP associated with the disease phenotype. When subjected to poly(A) tail-length assays, mitochondrial mRNAs from affected individuals were shown to have severely truncated poly(A) tails. Although defective mitochondrial DNA maintenance underlies a well-described group of clinical disorders, our findings reveal a defect of mitochondrial mRNA maturation associated with human disease and imply that this disease mechanism should be considered in other complex neurodegenerative disorders.

Functional roles of bestrophins in ocular epithelia.

There are four members of the bestrophin family of proteins in the human genome, of which two are known to be expressed in the eye. The gene BEST1 (formerly VMD2) which encodes the protein bestrophin-1 (Best1) was first identified in 1998. Mutations in this gene have now been associated with four clinically distinguishable human eye diseases, collectively referred to as "bestrophinopathies". Over the last decade, laboratories have sought to understand how Best1 mutations could result in eye diseases that range in presentation from macular degeneration to nanophthalmos. The majority of our knowledge comes from studies that have sought to understand how Best1 mutations or dysfunction could induce the classical symptoms of the most common of these diseases: Best vitelliform macular dystrophy (BVMD). BVMD is a dominant trait that is characterized electrophysiologically by a diminished electrooculogram light peak with a normal clinical electroretinogram. This together with the localization of Best1 to the retinal pigment epithelium (RPE) basolateral plasma membrane and data from heterologous expression studies, have led to the proposal that Best1 generates the light peak, and that bestrophins are a family of Ca(2+) activated Cl(-) channels (CaCCs). However, data from Best1 knock-out and knock-in mice, coupled with the recent discovery of a recessive bestrophinopathy suggest that Best1 does not generate the light peak. Recently Best2 was found to be expressed in non-pigmented epithelia in the ciliary body. However, aqueous dynamics in Best2 knock-out mice do not support a role for Best2 as a Cl(-) channel. Thus, the purported CaCC function of the bestrophins and how loss of this function relates to clinical disease needs to be reassessed. In this article, we examine data obtained from tissue-type and animal models and discuss the current state of bestrophin research, what roles Best1 and Best2 may play in ocular epithelia and ocular electrophysiology, and how perturbation of these functions may result in disease.

Lethal cystic kidney disease in Amish neonates associated with homozygous nonsense mutation of NPHP3.

BACKGROUND: Nephronophthisis is a group of genetically heterogeneous autosomal recessive cystic kidney disorders with a wide spectrum of severity and age of onset. We present a clinical and genetic study of a lethal form of nephronophthisis in neonates. STUDY DESIGN: Clinical and genetic investigations of a case series. SETTING & PARTICIPANTS: 12 affected offspring born to consanguineous parents from the Old Order Amish community. OUTCOMES: In this extended pedigree, the disorder is particularly severe; affected individuals survive only hours or days, with the cause of death invariably respiratory distress. RESULTS: Cystic kidneys were confirmed in 11 infants and suspected in an additional individual who had 2 affected siblings. Although the renal aspect of the phenotype was a consistent feature in all affected individuals, additional pulmonary, cardiac, and urinary tract abnormalities are variable parts of this syndrome. Physical mapping of the causative mutation in this extended Amish pedigree highlighted a 475-kilobase candidate region on chromosome 3 that contains the NPHP3 gene. Sequence analysis of this gene showed a cytosine to thymine substitution in exon 15 (c.2104C-->T) that cosegregated with the disease status. This substitution is predicted to lead to premature termination at position 702 of the protein product (p.Arg702X). LIMITATIONS: Because of the severe nature of this disease, few affected infants underwent full clinical evaluation. CONCLUSION: The presence of congenital malformations in the case series confirms the crucial role of NPHP3 in early embryonic development of the kidneys and urinary tract. The study also highlights the subtle variations in phenotypic expression in a cohort of patients with the same mutation in NPHP3.