Bi-allelic Loss-of-Function Mutations in the NPR-C Receptor Result in Enhanced Growth and Connective Tissue Abnormalities.

The natriuretic peptide signaling pathway has been implicated in many cellular processes, including endochondral ossification and bone growth. More precisely, different mutations in the NPR-B receptor and the CNP ligand have been identified in individuals with either short or tall stature. In this study we show that the NPR-C receptor (encoded by NPR3) is also important for the regulation of linear bone growth. We report four individuals, originating from three different families, with a phenotype characterized by tall stature, long digits, and extra epiphyses in the hands and feet. In addition, aortic dilatation was observed in two of these families. In each affected individual, we identified a bi-allelic loss-of-function mutation in NPR3. The missense mutations (c.442T>C [p.Ser148Pro] and c.1088A>T [p.Asp363Val]) resulted in intracellular retention of the NPR-C receptor and absent localization on the plasma membrane, whereas the nonsense mutation (c.1524delC [p.Tyr508∗]) resulted in nonsense-mediated mRNA decay. Biochemical analysis of plasma from two affected and unrelated individuals revealed a reduced NTproNP/NP ratio for all ligands and also high cGMP levels. These data strongly suggest a reduced clearance of natriuretic peptides by the defective NPR-C receptor and consequently increased activity of the NPR-A/B receptors. In conclusion, this study demonstrates that loss-of-function mutations in NPR3 result in increased NPR-A/B signaling activity and cause a phenotype marked by enhanced bone growth and cardiovascular abnormalities.

Sudden Cardiac Death Due to Deficiency of the Mitochondrial Inorganic Pyrophosphatase PPA2.

We have used whole-exome sequencing in ten individuals from four unrelated pedigrees to identify biallelic missense mutations in the nuclear-encoded mitochondrial inorganic pyrophosphatase (PPA2) that are associated with mitochondrial disease. These individuals show a range of severity, indicating that PPA2 mutations may cause a spectrum of mitochondrial disease phenotypes. Severe symptoms include seizures, lactic acidosis, cardiac arrhythmia, and death within days of birth. In the index family, presentation was milder and manifested as cardiac fibrosis and an exquisite sensitivity to alcohol, leading to sudden arrhythmic cardiac death in the second decade of life. Comparison of normal and mutant PPA2-containing mitochondria from fibroblasts showed that the activity of inorganic pyrophosphatase was significantly reduced in affected individuals. Recombinant PPA2 enzymes modeling hypomorphic missense mutations had decreased activity that correlated with disease severity. These findings confirm the pathogenicity of PPA2 mutations and suggest that PPA2 is a cardiomyopathy-associated protein, which has a greater physiological importance in mitochondrial function than previously recognized.

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.

SAMS, a syndrome of short stature, auditory-canal atresia, mandibular hypoplasia, and skeletal abnormalities is a unique neurocristopathy caused by mutations in Goosecoid.

Short stature, auditory canal atresia, mandibular hypoplasia, and skeletal abnormalities (SAMS) has been reported previously to be a rare, autosomal-recessive developmental disorder with other, unique rhizomelic skeletal anomalies. These include bilateral humeral hypoplasia, humeroscapular synostosis, pelvic abnormalities, and proximal defects of the femora. To identify the genetic basis of SAMS, we used molecular karyotyping and whole-exome sequencing (WES) to study small, unrelated families. Filtering of variants from the WES data included segregation analysis followed by comparison of in-house exomes. We identified a homozygous 306 kb microdeletion and homozygous predicted null mutations of GSC, encoding Goosecoid homeobox protein, a paired-like homeodomain transcription factor. This confirms that SAMS is a human malformation syndrome resulting from GSC mutations. Previously, Goosecoid has been shown to be a determinant at the Xenopus gastrula organizer region and a segment-polarity determinant in Drosophila. In the present report, we present data on Goosecoid protein localization in staged mouse embryos. These data and the SAMS clinical phenotype both suggest that Goosecoid is a downstream effector of the regulatory networks that define neural-crest cell-fate specification and subsequent mesoderm cell lineages in mammals, particularly during shoulder and hip formation. Our findings confirm that Goosecoid has an essential role in human craniofacial and joint development and suggest that Goosecoid is an essential regulator of mesodermal patterning in mammals and that it has specific functions in neural crest cell derivatives.

Enhanced diagnostic yield in Meckel-Gruber and Joubert syndrome through exome sequencing supplemented with split-read mapping.

BACKGROUND: The widespread adoption of high-throughput sequencing technologies by genetic diagnostic laboratories has enabled significant expansion of their testing portfolios. Rare autosomal recessive conditions have been a particular focus of many new services. Here we report a cohort of 26 patients referred for genetic analysis of Joubert (JBTS) and Meckel-Gruber (MKS) syndromes, two clinically and genetically heterogeneous neurodevelopmental conditions that define a phenotypic spectrum, with MKS at the severe end. METHODS: Exome sequencing was performed for all cases, using Agilent SureSelect v5 reagents and Illumina paired-end sequencing. For two cases medium-coverage (9×) whole genome sequencing was subsequently undertaken. RESULTS: Using a standard analysis pipeline for the detection of single nucleotide and small insertion or deletion variants, molecular diagnoses were confirmed in 12 cases (4%). Seeking to determine whether our cohort harboured pathogenic copy number variants (CNV), in JBTS- or MKS-associated genes, targeted comparative read-depth analysis was performed using FishingCNV. These analyses identified a putative intragenic AHI1 deletion that included three exons spanning at least 3.4 kb and an intergenic MPP4 to TMEM237 deletion that included exons spanning at least 21.5 kb. Whole genome sequencing enabled confirmation of the deletion-containing alleles and precise characterisation of the mutation breakpoints at nucleotide resolution. These data were validated following development of PCR-based assays that could be subsequently used for "cascade" screening and/or prenatal diagnosis. CONCLUSIONS: Our investigations expand the AHI1 and TMEM237 mutation spectrum and highlight the importance of performing CNV screening of disease-associated genes. We demonstrate a robust increasingly cost-effective CNV detection workflow that is applicable to all MKS/JBTS referrals.

Mutations in different components of FGF signaling in LADD syndrome.

Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by lacrimal duct aplasia, malformed ears and deafness, small teeth and digital anomalies. We identified heterozygous mutations in the tyrosine kinase domains of the genes encoding fibroblast growth factor receptors 2 and 3 (FGFR2, FGFR3) in LADD families, and in one further LADD family, we detected a mutation in the gene encoding fibroblast growth factor 10 (FGF10), a known FGFR ligand. These findings increase the spectrum of anomalies associated with abnormal FGF signaling.

The ETFDH c.158A>G variation disrupts the balanced interplay of ESE- and ESS-binding proteins thereby causing missplicing and multiple Acyl-CoA dehydrogenation deficiency.

Multiple acyl-CoA dehydrogenation deficiency is a disorder of fatty acid and amino acid oxidation caused by defects of electron transfer flavoprotein (ETF) or its dehydrogenase (ETFDH). A clear relationship between genotype and phenotype makes genotyping of patients important not only diagnostically but also for prognosis and for assessment of treatment. In the present study, we show that a predicted benign ETFDH missense variation (c.158A>G/p.Lys53Arg) in exon 2 causes exon skipping and degradation of ETFDH protein in patient samples. Using splicing reporter minigenes and RNA pull-down of nuclear proteins, we show that the c.158A>G variation increases the strength of a preexisting exonic splicing silencer (ESS) motif UAGGGA. This ESS motif binds splice inhibitory hnRNP A1, hnRNP A2/B1, and hnRNP H proteins. Binding of these inhibitory proteins prevents binding of the positive splicing regulatory SRSF1 and SRSF5 proteins to nearby and overlapping exonic splicing enhancer elements and this causes exon skipping. We further suggest that binding of hnRNP proteins to UAGGGA is increased by triggering synergistic hnRNP H binding to GGG triplets located upstream and downsteam of the UAGGGA motif. A number of disease-causing exonic elements that induce exon skipping in other genes have a similar architecture as the one in ETFDH exon 2.

An unusual phenotype of X-linked developmental delay and extreme behavioral difficulties associated with a mutation in the EBP gene.

We report on a family in which four males over three generations are affected with X-linked recessive developmental delay, learning difficulties, severe behavioral difficulties and mild dysmorphic features. Plasma sterol analysis in three of the four affected males demonstrated increased concentrations of 8-dehydrocholesterol (8-DHC) and cholest-8(9)-enol. All four affected males had a novel hemizygous missense mutation, p.W47R (c.139T>C), in EBP. Functional studies showed raised levels of cholest-8(9)-enol in patient's cultured fibroblast cells, which were suppressed when the cells were incubated with simvastatin. EBP encodes 3ß-hydroxysteroid-delta8, delta7-isomerase, a key enzyme involved in the cholesterol biosynthesis pathway. Mutations in EBP have previously been associated with Conradi-Hunermann-Happle syndrome (CHH), an X-linked dominant disorder characterized by skeletal dysplasia, skin, and ocular abnormalities, which is usually lethal in males. Four previous reports describe X-linked recessive multiple anomaly syndromes associated with non-mosaic EBP mutations in males, two at the same amino acid position, p.W47C. This phenotype has previously been described as "MEND" syndrome (male EBP disorder with neurological defects). The family reported herein represent either a novel phenotype, or an expansion of the MEND phenotype, characterized by extreme behavioral difficulties and a scarcity of structural anomalies. Simvastatin therapy is being evaluated in two males from this family.

Epilepsy, ataxia, sensorineural deafness, tubulopathy, and KCNJ10 mutations.

BACKGROUND: Five children from two consanguineous families presented with epilepsy beginning in infancy and severe ataxia, moderate sensorineural deafness, and a renal salt-losing tubulopathy with normotensive hypokalemic metabolic alkalosis. We investigated the genetic basis of this autosomal recessive disease, which we call the EAST syndrome (the presence of epilepsy, ataxia, sensorineural deafness, and tubulopathy). METHODS: Whole-genome linkage analysis was performed in the four affected children in one of the families. Newly identified mutations in a potassium-channel gene were evaluated with the use of a heterologous expression system. Protein expression and function were further investigated in genetically modified mice. RESULTS: Linkage analysis identified a single significant locus on chromosome 1q23.2 with a lod score of 4.98. This region contained the KCNJ10 gene, which encodes a potassium channel expressed in the brain, inner ear, and kidney. Sequencing of this candidate gene revealed homozygous missense mutations in affected persons in both families. These mutations, when expressed heterologously in xenopus oocytes, caused significant and specific decreases in potassium currents. Mice with Kcnj10 deletions became dehydrated, with definitive evidence of renal salt wasting. CONCLUSIONS: Mutations in KCNJ10 cause a specific disorder, consisting of epilepsy, ataxia, sensorineural deafness, and tubulopathy. Our findings indicate that KCNJ10 plays a major role in renal salt handling and, hence, possibly also in blood-pressure maintenance and its regulation.

Altered electroretinograms in patients with KCNJ10 mutations and EAST syndrome.

The K+ channel expressed by the KCNJ10 gene (Kir4.1) has previously demonstrated importance in retinal function in animal experiments. Recently, mutations in KCNJ10 were recognised as pathogenic in man, causing a constellation of symptoms, including epilepsy, ataxia, sensorineural deafness and a renal tubulopathy designated as EAST syndrome. We have studied the impact of KCNJ10 mutations on the human electroretinogram (ERG) in four unrelated patients with EAST syndrome. Corneal ganzfeld ERGs were elicited in response to flash stimuli of strengths of 0.001­10 phot cd s/m2 presented scotopically, and 0.3­10 phot cd s/m2 presented photopically. ERG waveforms from light-adapted retinae of all patients showed reduced amplitudes of the photopic negative response (PhNR) (P < 0.001). The photopic ERGs showed a delay in b-wave time to peak, but the photopic hill, i.e. the relative variation of time to peak and amplitude with luminance flash strength, was preserved. Scotopic ERGs to flash strengths 0.01 to 0.1 phot cd s/m2 showed a delay of up to 20 ms before the onset of the b-wave in two patients compared to controls. Stimulus­response functions were fitted by Michaelis­Menten equations and showed significantly lower retinal sensitivity in two patients than in controls (P < 0.001). Our study for the first time in the human ERG shows changes in association with KCNJ10 mutations affecting a Muller cell K+ channel. These data illustrate the role of KCNJ10 function in the physiology of proximal and possibly also the distal human retina.

Clinical characterization of a newly described neonatal diabetes syndrome caused by RFX6 mutations.

Mutations in the RFX6 gene were recently described to underlie a distinct autosomal recessive syndrome of neonatal diabetes comprising intestinal atresia and hepatobiliary abnormalities. Until now, only six patients harboring RFX6 mutations have been reported. We report on a new case due to a novel homozygous splice site mutation and update on the clinical outcome of a previously reported patient. In addition we review the clinical and molecular features of all RFX6 mutated cases to better characterize the syndrome. Our results suggest that despite the early postnatal fulminant course, patients who survive may expect a relatively favorable prognosis.

cGAS-mediated induction of type I interferon due to inborn errors of histone pre-mRNA processing.

Inappropriate stimulation or defective negative regulation of the type I interferon response can lead to autoinflammation. In genetically uncharacterized cases of the type I interferonopathy Aicardi-Goutières syndrome, we identified biallelic mutations in LSM11 and RNU7-1, which encode components of the replication-dependent histone pre-mRNA-processing complex. Mutations were associated with the misprocessing of canonical histone transcripts and a disturbance of linker histone stoichiometry. Additionally, we observed an altered distribution of nuclear cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and enhanced interferon signaling mediated by the cGAS-stimulator of interferon genes (STING) pathway in patient-derived fibroblasts. Finally, we established that chromatin without linker histone stimulates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) production in vitro more efficiently. We conclude that nuclear histones, as key constituents of chromatin, are essential in suppressing the immunogenicity of self-DNA.

Mutational, functional, and expression studies of the TCF4 gene in Pitt-Hopkins syndrome.

Pitt-Hopkins syndrome is a severe congenital encephalopathy recently ascribed to de novo heterozygous TCF4 gene mutations. We report a series of 13 novel PHS cases with a TCF4 mutation and show that EEG, brain magnetic resonance imagain (MRI), and immunological investigations provide valuable additional clues to the diagnosis. We confirm a mutational hot spot in the basic domain of the E-protein. Functional studies illustrate that heterodimerisation of mutant TCF4 proteins with a tissue-specific transcription factor is less effective than that homodimerisation in a luciferase reporter assay. We also show that the TCF4 expression pattern in human embryonic development is widespread but not ubiquitous. In summary, we further delineate an underdiagnosed mental retardation syndrome, highlighting TCF4 function during development and facilitating diagnosis within the first year of life.

A further example of a distinctive autosomal recessive syndrome comprising neonatal diabetes mellitus, intestinal atresias and gall bladder agenesis.

We report a patient born to consanguineous parents as a further example of a recently described phenotype comprising neonatal diabetes, intestinal atresias and gall bladder agenesis. Other reports have described cases with overlapping patterns including malrotation, biliary atresia and pancreatic hypoplasia (e.g. as described by Martínez-Frías). We propose that these cases may represent variations of the same syndrome. It is likely that this disorder is inherited as an autosomal recessive trait. Our case is the first to have neonatal diabetes without a demonstrable structural pancreatic abnormality, showing that a deficit in pancreatic function is involved. We sequenced genes with a recognized role in monogenic forms of diabetes, including KCNJ11, ABCC8, GCK, IPF1, HNF1beta, NeuroD1 and TCF7L2, as well as a novel candidate gene, HNF6, known to be involved in hepatobiliary and pancreatic development, but did not identify mutations.

Brachydactyly with extrahepatic biliary atresia, patent ductus arteriosus and seizures--a new syndrome?

Brachydactyly is a relatively common congenital anomaly and can be associated with many other malformations. However, brachydactyly in association with biliary atresia is rare. We present a male child with strikingly symmetrical brachydactyly and nail hypoplasia, extrahepatic biliary atresia, patent ductus arteriosus, seizures, developmental delay and cataracts. This combination of features has not previously been described and we suggest that this case represents a new syndrome.

Study of 250 children with idiopathic mental retardation reveals nine cryptic and diverse subtelomeric chromosome anomalies.

Cryptic subtelomeric chromosome anomalies have been recognized as a significant cause of dysmorphology and mental retardation. To determine whether the clinical cytogenetics laboratory should screen routinely for these aberrations, we have tested 250 patients with idiopathic mental retardation/developmental delay, either isolated (53) or associated with dysmorphic features and/or malformations in the absence of a recognizable syndrome (197). All had normal karyotypes at the 550-850 band level. Subtelomeric anomalies were found in 1/53 of the first group (1.9%) and 8/197 of the second group (4.1%). In one patient, two separate anomalies were present: a deletion (not inherited) and a duplication (inherited). It is possible that one of these 10 observed aberrations might represent a rare and previously unreported polymorphism and one a rare cross-hybridization. Our study supports the proposition that cryptic subtelomeric rearrangements are a significant cause of idiopathic mental retardation/developmental delay, but both the diversity of the phenotypes of the positive cases and the wide diversity of their associated chromosome abnormalities emphasize the central problem for the clinical cytogenetics laboratory-that of choosing the most productive patient base for this useful diagnostic test.

Macrocephaly and sclerosis of the tubular bones in an isolated patient: a mild case of craniodiaphyseal dysplasia?

We report a 56-year-old woman, mainly suffering from painful legs and the inability to run. Radiologically, marked sclerosis and hyperostosis of the skull bones is present resulting in macrocephaly. Most tubular bones of the limbs, as well as the clavicles, are affected by sclerosis. By mutation analysis of the TGFB1, SOST and LRP5 genes, we were able to exclude the diagnoses of Camurati-Engelmann disease, Van Buchem disease, sclerosteosis, high-bone-mass trait and endosteal hyperostosis (Worth type). We believe this patient represents one of the very few examples of adult craniodiaphyseal dysplasia with a mild form of the disease and moderate facial changes.

SURF1 deficiency: a multi-centre natural history study.

BACKGROUND: SURF1 deficiency, a monogenic mitochondrial disorder, is the most frequent cause of cytochrome c oxidase (COX) deficient Leigh syndrome (LS). We report the first natural history study of SURF1 deficiency. METHODS: We conducted a multi-centre case notes review of 44 SURF1-deficient patients from ten different UK centres and two Australian centres. Survival data for LRPPRC-deficient LS and nuclear-encoded complex I-deficient LS patients were obtained from previous publications. The survival of SURF1-deficient patients was compared with these two groups using Kaplan-Meier survival analysis and logrank test. RESULTS: The majority of patients (32/44, 73%) presented in infancy (median 9.5 months). Frequent symptoms were poor weight gain (95%, median age 10 months), hypotonia (93%, median age 14 months), poor feeding/vomiting (89%, median age 10 months), developmental delay (88%, median age 14 months), developmental regression (71%, median age 19 months), movement disorder (52%, median age 24 months), oculomotor involvement (52%, median age 29 months) and central respiratory failure (78%, median age 31 months). Hypertrichosis (41%), optic atrophy (23%), encephalopathy (20%), seizures (14%) and cardiomyopathy (2%) were observed less frequently. CONCLUSIONS: SURF1-deficient patients have a homogeneous clinical and biochemical phenotype. Early recognition is essential to expedite diagnosis and enable prenatal diagnosis.

Rapid visualisation of microarray copy number data for the detection of structural variations linked to a disease phenotype.

Whilst the majority of inherited diseases have been found to be caused by single base substitutions, small insertions or deletions (<1Kb), a significant proportion of genetic variability is due to copy number variation (CNV). The possible role of CNV in monogenic and complex diseases has recently attracted considerable interest. However, until the development of whole genome, oligonucleotide micro-arrays, designed specifically to detect the presence of copy number variation, it was not easy to screen an individual for the presence of unknown deletions or duplications with sizes below the level of sensitivity of optical microscopy (3-5 Mb). Now that currently available oligonucleotide micro-arrays have in excess of a million probes, the problem of copy number analysis has moved from one of data production to that of data analysis. We have developed CNViewer, to identify copy number variation that co-segregates with a disease phenotype in small nuclear families, from genome-wide oligonucleotide micro-array data. This freely available program should constitute a useful addition to the diagnostic armamentarium of clinical geneticists.