AXIN1 bi-allelic variants disrupting the C-terminal DIX domain cause craniometadiaphyseal osteosclerosis with hip dysplasia.

Sclerosing skeletal dysplasias result from an imbalance between bone formation and resorption. We identified three homozygous, C-terminally truncating AXIN1 variants in seven individuals from four families affected by macrocephaly, cranial hyperostosis, and vertebral endplate sclerosis. Other frequent findings included hip dysplasia, heart malformations, variable developmental delay, and hematological anomalies. In line with AXIN1 being a central component of the ß-catenin destruction complex, analyses of primary and genome-edited cells harboring the truncating variants revealed enhanced basal canonical Wnt pathway activity. All three AXIN1-truncating variants resulted in reduced protein levels and impaired AXIN1 polymerization mediated by its C-terminal DIX domain but partially retained Wnt-inhibitory function upon overexpression. Addition of a tankyrase inhibitor attenuated Wnt overactivity in the AXIN1-mutant model systems. Our data suggest that AXIN1 coordinates the action of osteoblasts and osteoclasts and that tankyrase inhibitors can attenuate the effects of AXIN1 hypomorphic variants.

Pathogenic variants of sphingomyelin synthase SMS2 disrupt lipid landscapes in the secretory pathway.

Sphingomyelin is a dominant sphingolipid in mammalian cells. Its production in the trans-Golgi traps cholesterol synthesized in the ER to promote formation of a sphingomyelin/sterol gradient along the secretory pathway. This gradient marks a fundamental transition in physical membrane properties that help specify organelle identify and function. We previously identified mutations in sphingomyelin synthase SMS2 that cause osteoporosis and skeletal dysplasia. Here, we show that SMS2 variants linked to the most severe bone phenotypes retain full enzymatic activity but fail to leave the ER owing to a defective autonomous ER export signal. Cells harboring pathogenic SMS2 variants accumulate sphingomyelin in the ER and display a disrupted transbilayer sphingomyelin asymmetry. These aberrant sphingomyelin distributions also occur in patient-derived fibroblasts and are accompanied by imbalances in cholesterol organization, glycerophospholipid profiles, and lipid order in the secretory pathway. We postulate that pathogenic SMS2 variants undermine the capacity of osteogenic cells to uphold nonrandom lipid distributions that are critical for their bone forming activity.

The MAP3K7 gene: Further delineation of clinical characteristics and genotype/phenotype correlations.

Mitogen-activated protein 3 kinase 7 (MAP3K7) encodes the ubiquitously expressed transforming growth factor ß-activated kinase 1, which plays a crucial role in many cellular processes. Mutationsin the MAP3K7 gene have been linked to two distinct disorders: frontometaphyseal dysplasia type 2 (FMD2) and cardiospondylocarpofacial syndrome (CSCF). The fact that different mutations can induce two distinct phenotypes suggests a phenotype/genotype correlation, but no side-by-side comparison has been done thus far to confirm this. Here, we significantly expand the cohort and the description of clinical phenotypes for patients with CSCF and FMD2 who carry mutations in MAP3K7. Our findings support that in contrast to FMD2-causing mutations, CSCF-causing mutations in MAP3K7 have a loss-of-function effect. Additionally, patients with pathogenic mutations in MAP3K7 are at risk for (severe) cardiac disease, have symptoms associated with connective tissue disease, and we show overlap in clinical phenotypes of CSCF with Noonan syndrome (NS). Together, we confirm a molecular fingerprint of FMD2- versus CSCF-causing MAP3K7 mutations and conclude that mutations in MAP3K7 should be considered in the differential diagnosis of patients with syndromic congenital cardiac defects and/or cardiomyopathy, syndromic connective tissue disorders, and in the differential diagnosis of NS.

Biallelic variants in POLR3GL cause endosteal hyperostosis and oligodontia.

RNA polymerase III (Pol III) is an essential 17-subunit complex responsible for the transcription of small housekeeping RNAs such as transfer RNAs and 5S ribosomal RNA. Biallelic variants in four genes (POLR3A, POLR3B, and POLR1C and POLR3K) encoding Pol III subunits have previously been found in individuals with (neuro-) developmental disorders. In this report, we describe three individuals with biallelic variants in POLR3GL, a gene encoding a Pol III subunit that has not been associated with disease before. Using whole exome sequencing in a monozygotic twin and an unrelated individual, we detected homozygous and compound heterozygous POLR3GL splice acceptor site variants. RNA sequencing confirmed the loss of full-length POLR3GL RNA transcripts in blood samples of the individuals. The phenotypes of the described individuals are mainly characterized by axial endosteal hyperostosis, oligodontia, short stature, and mild facial dysmorphisms. These features largely fit within the spectrum of phenotypes caused by previously described biallelic variants in POLR3A, POLR3B, POLR1C, and POLR3K. These findings further expand the spectrum of POLR3-related disorders and implicate that POLR3GL should be included in genetic testing if such disorders are suspected.

Osteoporosis and skeletal dysplasia caused by pathogenic variants in SGMS2.

Mechanisms leading to osteoporosis are incompletely understood. Genetic disorders with skeletal fragility provide insight into metabolic pathways contributing to bone strength. We evaluated 6 families with rare skeletal phenotypes and osteoporosis by next-generation sequencing. In all the families, we identified a heterozygous variant in SGMS2, a gene prominently expressed in cortical bone and encoding the plasma membrane-resident sphingomyelin synthase SMS2. Four unrelated families shared the same nonsense variant, c.148C>T (p.Arg50*), whereas the other families had a missense variant, c.185T>G (p.Ile62Ser) or c.191T>G (p.Met64Arg). Subjects with p.Arg50* presented with childhood-onset osteoporosis with or without cranial sclerosis. Patients with p.Ile62Ser or p.Met64Arg had a more severe presentation, with neonatal fractures, severe short stature, and spondylometaphyseal dysplasia. Several subjects had experienced peripheral facial nerve palsy or other neurological manifestations. Bone biopsies showed markedly altered bone material characteristics, including defective bone mineralization. Osteoclast formation and function in vitro was normal. While the p.Arg50* mutation yielded a catalytically inactive enzyme, p.Ile62Ser and p.Met64Arg each enhanced the rate of de novo sphingomyelin production by blocking export of a functional enzyme from the endoplasmic reticulum. SGMS2 pathogenic variants underlie a spectrum of skeletal conditions, ranging from isolated osteoporosis to complex skeletal dysplasia, suggesting a critical role for plasma membrane-bound sphingomyelin metabolism in skeletal homeostasis.

BCL11B mutations in patients affected by a neurodevelopmental disorder with reduced type 2 innate lymphoid cells.

The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.

Two novel cases expanding the phenotype of SETD2-related overgrowth syndrome.

The SETD2-related overgrowth syndrome is also called "Luscan-Lumish syndrome" (OMIM 616831) with the clinical characteristics of intellectual disability, speech delay, macrocephaly, facial dysmorphism, and autism spectrum disorders. We report on two novel patients a 4.5-year-old boy and a 23-year-old female adolescent with a speech and language developmental delay, autism spectrum disorder and macrocephaly, who were both diagnosed with SETD2-related overgrowth syndrome due to de novo frameshift mutations in the SETD2 gene. Features not previously described which were present in either one of our patients were nasal polyps, a large tongue with creases, a high pain threshold, constipation, and undescended testicles. These features may be related to the syndrome and may need special attention in future patients. Additionally, prevention of obesity should be an important point of attention for patients diagnosed with a SETD2-related overgrowth syndrome.

Further delineation of the GDF6 related multiple synostoses syndrome.

A mutation in GDF6 was recently found to underlie a multiple synostoses syndrome. In this report, we describe the second family with GDF6-related multiple synostoses syndrome (SYNS4), caused by a novel c.1287C>A/p.Ser429Arg mutation in GDF6. In addition to synostoses of carpal and/or tarsal bones, at least 6 of 10 affected patients in this family have been diagnosed with mild to moderate hearing loss. In four of them otosclerosis was said to be present, one patient had hearing loss due to severe stapes fixation at the age of 6 years, providing evidence that hearing loss in the GDF6-related multiple synostoses syndrome can be present in childhood. Two others had surgery for stapes fixation at adult age. We hypothesize that, identical to the recently published GDF6-related multiple synostoses family, the p.Ser429Arg mutation also leads to a gain of function. The previously reported c.1330T>A/pTyr444Asn mutation was located in a predicted Noggin and receptor I interacting domain and the gain of function was partly due to resistance of the mutant GDF6 to the BMP-inhibitor Noggin. The results in our family show that mutations predicting to affect the type II receptor interface can lead to a similar phenotype and that otosclerosis presenting in childhood can be part of the GDF6-related multiple synostoses syndrome.

De Novo Mutations in CHD4, an ATP-Dependent Chromatin Remodeler Gene, Cause an Intellectual Disability Syndrome with Distinctive Dysmorphisms.

Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler involved in epigenetic regulation of gene transcription, DNA repair, and cell cycle progression. Also known as Mi2ß, CHD4 is an integral subunit of a well-characterized histone deacetylase complex. Here we report five individuals with de novo missense substitutions in CHD4 identified through whole-exome sequencing and web-based gene matching. These individuals have overlapping phenotypes including developmental delay, intellectual disability, hearing loss, macrocephaly, distinct facial dysmorphisms, palatal abnormalities, ventriculomegaly, and hypogonadism as well as additional findings such as bone fusions. The variants, c.3380G>A (p.Arg1127Gln), c.3443G>T (p.Trp1148Leu), c.3518G>T (p.Arg1173Leu), and c.3008G>A, (p.Gly1003Asp) (GenBank: NM_001273.3), affect evolutionarily highly conserved residues and are predicted to be deleterious. Previous studies in yeast showed the equivalent Arg1127 and Trp1148 residues to be crucial for SNF2 function. Furthermore, mutations in the same positions were reported in malignant tumors, and a de novo missense substitution in an equivalent arginine residue in the C-terminal helicase domain of SMARCA4 is associated with Coffin Siris syndrome. Cell-based studies of the p.Arg1127Gln and p.Arg1173Leu mutants demonstrate normal localization to the nucleus and HDAC1 interaction. Based on these findings, the mutations potentially alter the complex activity but not its formation. This report provides evidence for the role of CHD4 in human development and expands an increasingly recognized group of Mendelian disorders involving chromatin remodeling and modification.

Compound heterozygous NEK1 variants in two siblings with oral-facial-digital syndrome type II (Mohr syndrome).

The oral-facial-digital (OFD) syndromes comprise a group of related disorders with a combination of oral, facial and digital anomalies. Variants in several ciliary genes have been associated with subtypes of OFD syndrome, yet in most OFD patients the underlying cause remains unknown. We investigated the molecular basis of disease in two brothers with OFD type II, Mohr syndrome, by performing single-nucleotide polymorphism (SNP)-array analysis on the brothers and their healthy parents to identify homozygous regions and candidate genes. Subsequently, we performed whole-exome sequencing (WES) on the family. Using WES, we identified compound heterozygous variants c.[464G>C];[1226G>A] in NIMA (Never in Mitosis Gene A)-Related Kinase 1 (NEK1). The novel variant c.464G>C disturbs normal splicing in an essential region of the kinase domain. The nonsense variant c.1226G>A, p.(Trp409*), results in nonsense-associated alternative splicing, removing the first coiled-coil domain of NEK1. Candidate variants were confirmed with Sanger sequencing and alternative splicing assessed with cDNA analysis. Immunocytochemistry was used to assess cilia number and length. Patient-derived fibroblasts showed severely reduced ciliation compared with control fibroblasts (18.0 vs 48.9%, P<0.0001), but showed no significant difference in cilia length. In conclusion, we identified compound heterozygous deleterious variants in NEK1 in two brothers with Mohr syndrome. Ciliation in patient fibroblasts is drastically reduced, consistent with a ciliary defect pathogenesis. Our results establish NEK1 variants involved in the etiology of a subset of patients with OFD syndrome type II and support the consideration of including (routine) NEK1 analysis in patients suspected of OFD.

A study of the clinical and radiological features in a cohort of 93 patients with a COL2A1 mutation causing spondyloepiphyseal dysplasia congenita or a related phenotype.

Type 2 collagen disorders encompass a diverse group of skeletal dysplasias that are commonly associated with orthopedic, ocular, and hearing problems. However, the frequency of many clinical features has never been determined. We retrospectively investigated the clinical, radiological, and genotypic data in a group of 93 patients with molecularly confirmed SEDC or a related disorder. The majority of the patients (80/93) had short stature, with radiological features of SEDC (n = 64), others having SEMD (n = 5), Kniest dysplasia (n = 7), spondyloperipheral dysplasia (n = 2), or Torrance-like dysplasia (n = 2). The remaining 13 patients had normal stature with mild SED, Stickler-like syndrome or multiple epiphyseal dysplasia. Over 50% of the patients had undergone orthopedic surgery, usually for scoliosis, femoral osteotomy or hip replacement. Odontoid hypoplasia was present in 56% (95% CI 38-74) and a correlation between odontoid hypoplasia and short stature was observed. Atlanto-axial instability, was observed in 5 of the 18 patients (28%, 95% CI 10-54) in whom flexion-extension films of the cervical spine were available; however, it was rarely accompanied by myelopathy. Myopia was found in 45% (95% CI 35-56), and retinal detachment had occurred in 12% (95% CI 6-21; median age 14 years; youngest age 3.5 years). Thirty-two patients complained of hearing loss (37%, 95% CI 27-48) of whom 17 required hearing aids. The ophthalmological features and possibly also hearing loss are often relatively frequent and severe in patients with splicing mutations. Based on clinical findings, age at onset and genotype-phenotype correlations in this cohort, we propose guidelines for the management and follow-up in this group of disorders.

The IFITM5 mutation c.-14C > T results in an elongated transcript expressed in human bone; and causes varying phenotypic severity of osteogenesis imperfecta type V.

BACKGROUND: The genetic mutation resulting in osteogenesis imperfecta (OI) type V was recently characterised as a single point mutation (c.-14C > T) in the 5' untranslated region (UTR) of IFITM5, a gene encoding a transmembrane protein with expression restricted to skeletal tissue. This mutation creates an alternative start codon and has been shown in a eukaryotic cell line to result in a longer variant of IFITM5, but its expression has not previously been demonstrated in bone from a patient with OI type V. METHODS: Sanger sequencing of the IFITM5 5' UTR was performed in our cohort of subjects with a clinical diagnosis of OI type V. Clinical data was collated from referring clinicians. RNA was extracted from a bone sample from one patient and Sanger sequenced to determine expression of wild-type and mutant IFITM5. RESULTS: All nine subjects with OI type V were heterozygous for the c.-14C > T IFITM5 mutation. Clinically, there was heterogeneity in phenotype, particularly in the manifestation of bone fragility amongst subjects. Both wild-type and mutant IFITM5 mRNA transcripts were present in bone. CONCLUSIONS: The c.-14C > T IFITM5 mutation does not result in an RNA-null allele but is expressed in bone. Individuals with identical mutations in IFITM5 have highly variable phenotypic expression, even within the same family.

[Multiple epiphyseal dysplasia: skeletal dysplasia presenting as neuromuscular disease]. / Multipele epifysaire dysplasie: een skeletaandoening met een neuromusculaire presentatie.

BACKGROUND: Multiple epiphyseal dysplasia (MED) is a clinically and genetically heterogeneous skeletal dysplasia. MED is usually inherited as a dominant trait, however, in a quarter of patients it is a recessive trait. Common symptoms of MED are pain and stiffness of joints. However, MED also can present as a neuromuscular disorder with hypotonia and muscle weakness, particularly in childhood. CASE DESCRIPTION: We saw a 7-year-old boy with abnormal gait and slight weakness of the upper legs at our outpatient clinic. Because of his short stature and other skeletal anomalies, skeletal dysplasia was considered. The diagnosis of MED was confirmed by DNA analysis. A mutation in the cartilage oligomeric matrix protein (COMP) gene was found. CONCLUSION: Skeletal dysplasia should be included in the differential diagnosis of children with unexplained neuromuscular symptoms.

Mutations in genes encoding the cadherin receptor-ligand pair DCHS1 and FAT4 disrupt cerebral cortical development.

The regulated proliferation and differentiation of neural stem cells before the generation and migration of neurons in the cerebral cortex are central aspects of mammalian development. Periventricular neuronal heterotopia, a specific form of mislocalization of cortical neurons, can arise from neuronal progenitors that fail to negotiate aspects of these developmental processes. Here we show that mutations in genes encoding the receptor-ligand cadherin pair DCHS1 and FAT4 lead to a recessive syndrome in humans that includes periventricular neuronal heterotopia. Reducing the expression of Dchs1 or Fat4 within mouse embryonic neuroepithelium increased progenitor cell numbers and reduced their differentiation into neurons, resulting in the heterotopic accumulation of cells below the neuronal layers in the neocortex, reminiscent of the human phenotype. These effects were countered by concurrent knockdown of Yap, a transcriptional effector of the Hippo signaling pathway. These findings implicate Dchs1 and Fat4 upstream of Yap as key regulators of mammalian neurogenesis.

X-exome sequencing identifies a HDAC8 variant in a large pedigree with X-linked intellectual disability, truncal obesity, gynaecomastia, hypogonadism and unusual face.

BACKGROUND: We present a large Dutch family with seven males affected by a novel syndrome of X-linked intellectual disability, hypogonadism, gynaecomastia, truncal obesity, short stature and recognisable craniofacial manifestations resembling but not identical to Wilson-Turner syndrome. Seven female relatives show a much milder expression of the phenotype. METHODS AND RESULTS: We performed X chromosome exome (X-exome) sequencing in five individuals from this family and identified a novel intronic variant in the histone deacetylase 8 gene (HDAC8), c.164+5G>A, which disturbs the normal splicing of exon 2 resulting in exon skipping, and introduces a premature stop at the beginning of the histone deacetylase catalytic domain. The identified variant completely segregates in this family and was absent in 96 Dutch controls and available databases. Affected female carriers showed a notably skewed X-inactivation pattern in lymphocytes in which the mutated X-chromosome was completely inactivated. CONCLUSIONS: HDAC8 is a member of the protein family of histone deacetylases that play a major role in epigenetic gene silencing during development. HDAC8 specifically controls the patterning of the skull with the mouse HDAC8 knock-out showing craniofacial deformities of the skull. The present family provides the first evidence for involvement of HDAC8 in a syndromic form of intellectual disability.

Mutation-based growth charts for SEDC and other COL2A1 related dysplasias.

From data collected via a large international collaborative study, we have constructed a growth chart for patients with molecularly confirmed congenital spondylo-epiphyseal dysplasia (SEDC) and other COL2A1 related dysplasias. The growth chart is based on longitudinal height measurements of 79 patients with glycine substitutions in the triple-helical domain of COL2A1. In addition, measurements of 27 patients with other molecular defects, such as arginine to cysteine substitutions, splice mutations, and mutations in the C-terminal propeptide have been plotted on the chart. Height of the patients progressively deviate from that of normal children: compared to normal WHO charts, the mean length/height is -2.6 SD at birth, -4.2 SD at 5 years, and -5.8 SD in adulthood. The mean adult height (male and female combined) of patients with glycine substitutions in the triple-helical region is 138.2 cm but there is a large variation. Patients with glycine to cysteine substitutions tend to cluster within the upper part of the chart, while patients with glycine to serine or valine substitutions are situated between +1 SD and -1 SD. Patients with carboxy-terminal glycine substitutions tend to be shorter than patients with amino-terminal substitutions, while patients with splice mutations are relatively tall. However, there are exceptions and specific mutations can have a strong or a relatively mild negative effect on growth. The observation of significant difference in adult height between affected members of the same family indicates that height remains a multifactorial trait even in the presence of a mutation with a strong dominant effect.

Dominant missense mutations in ABCC9 cause Cantú syndrome.

Cantú syndrome is characterized by congenital hypertrichosis, distinctive facial features, osteochondrodysplasia and cardiac defects. By using family-based exome sequencing, we identified a de novo mutation in ABCC9. Subsequently, we discovered novel dominant missense mutations in ABCC9 in 14 of the 16 individuals with Cantú syndrome examined. The ABCC9 protein is part of an ATP-dependent potassium (K(ATP)) channel that couples the metabolic state of a cell with its electrical activity. All mutations altered amino acids in or close to the transmembrane domains of ABCC9. Using electrophysiological measurements, we show that mutations in ABCC9 reduce the ATP-mediated potassium channel inhibition, resulting in channel opening. Moreover, similarities between the phenotype of individuals with Cantú syndrome and side effects from the K(ATP) channel agonist minoxidil indicate that the mutations in ABCC9 result in channel opening. Given the availability of ABCC9 antagonists, our findings may have direct implications for the treatment of individuals with Cantú syndrome.

Multicentric carpotarsal osteolysis is caused by mutations clustering in the amino-terminal transcriptional activation domain of MAFB.

Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia characterized by aggressive osteolysis, particularly affecting the carpal and tarsal bones, and is frequently associated with progressive renal failure. Using exome capture and next-generation sequencing in five unrelated simplex cases of MCTO, we identified previously unreported missense mutations clustering within a 51 base pair region of the single exon of MAFB, validated by Sanger sequencing. A further six unrelated simplex cases with MCTO were also heterozygous for previously unreported mutations within this same region, as were affected members of two families with autosomal-dominant MCTO. MAFB encodes a transcription factor that negatively regulates RANKL-induced osteoclastogenesis and is essential for normal renal development. Identification of this gene paves the way for development of novel therapeutic approaches for this crippling disease and provides insight into normal bone and kidney development.

Unbalanced der(5)t(5;20) translocation associated with megalencephaly, perisylvian polymicrogyria, polydactyly and hydrocephalus.

The combination of megalencephaly, perisylvian polymicrogyria, polydactyly and hydrocephalus (MPPH) is a rare syndrome of unknown cause. We observed two first cousins affected by an MPPH-like phenotype with a submicroscopic chromosome 5q35 deletion as a result of an unbalanced der(5)t(5;20)(q35.2;q13.3) translocation, including the NSD1 Sotos syndrome locus. We describe the phenotype and the deletion breakpoints of the two MPPH-like patients and compare these with five unrelated MPPH and Sotos patients harboring a 5q35 microdeletion. Mapping of the breakpoints in the two cousins was performed by MLPA, FISH, high density SNP-arrays and Q-PCR for the 5q35 deletion and 20q13 duplication. The 5q35 deletion area of the two cousins almost completely overlaps with earlier described patients with an atypical Sotos microdeletion, except for the DRD1 gene. The five unrelated MPPH patients neither showed submicroscopic chromosomal aberrations nor DRD1 mutations. We reviewed the brain MRI of 10 Sotos patients and did not detect polymicrogyria in any of them. In our two cousins, the MPPH-like phenotype is probably caused by the contribution of genes on both chromosome 5q35 and 20q13. Some patients with MPPH may harbor a submicroscopic chromosomal aberration and therefore high-resolution array analysis should be part of the diagnostic workup.