Biallelic variants in PPP1R13L cause paediatric dilated cardiomyopathy.

Childhood dilated cardiomyopathy (DCM) is a leading cause of heart failure requiring cardiac transplantation and approximately 5% of cases result in sudden death. Knowledge of the underlying genetic cause can aid prognostication and clinical management and enables accurate recurrence risk counselling for the family. Here we used genomic sequencing to identify the causative genetic variant(s) in families with children affected by severe DCM. In an international collaborative effort facilitated by GeneMatcher, biallelic variants in PPP1R13L were identified in seven children with severe DCM from five unrelated families following exome or genome sequencing and inheritance-based variant filtering. PPP1R13L encodes inhibitor of apoptosis-stimulating protein of p53 protein (iASPP). In addition to roles in apoptosis, iASPP acts as a regulator of desmosomes and has been implicated in inflammatory pathways. DCM presented early (mean: 2 years 10 months; range: 3 months-9 years) and was progressive, resulting in death (n = 3) or transplant (n = 3), with one child currently awaiting transplant. Genomic sequencing technologies are valuable for the identification of novel and emerging candidate genes. Biallelic variants in PPP1R13L were previously reported in a single consanguineous family with paediatric DCM. The identification here of a further five families now provides sufficient evidence to support a robust gene-disease association between PPP1R13L and severe paediatric DCM. The PPP1R13L gene should be included in panel-based genetic testing for paediatric DCM.

NaV1.7 gain-of-function mutations as a continuum: A1632E displays physiological changes associated with erythromelalgia and paroxysmal extreme pain disorder mutations and produces symptoms of both disorders.

Gain-of-function mutations of Na(V)1.7 have been shown to produce two distinct disorders: Na(V)1.7 mutations that enhance activation produce inherited erythromelalgia (IEM), characterized by burning pain in the extremities; Na(V)1.7 mutations that impair inactivation produce a different, nonoverlapping syndrome, paroxysmal extreme pain disorder (PEPD), characterized by rectal, periocular, and perimandibular pain. Here we report a novel Na(V)1.7 mutation associated with a mixed clinical phenotype with characteristics of IEM and PEPD, with an alanine 1632 substitution by glutamate (A1632E) in domain IV S4-S5 linker. Patch-clamp analysis shows that A1632E produces changes in channel function seen in both IEM and PEPD mutations: A1632E hyperpolarizes (-7 mV) the voltage dependence of activation, slows deactivation, and enhances ramp responses, as observed in Na(V)1.7 mutations that produce IEM. A1632E depolarizes (+17mV) the voltage dependence of fast inactivation, slows fast inactivation, and prevents full inactivation, resulting in persistent inward currents similar to PEPD mutations. Using current clamp, we show that A1632E renders dorsal root ganglion (DRG) and trigeminal ganglion neurons hyperexcitable. These results demonstrate a Na(V)1.7 mutant with biophysical characteristics common to PEPD (impaired fast inactivation) and IEM (hyperpolarized activation, slow deactivation, and enhanced ramp currents) associated with a clinical phenotype with characteristics of both IEM and PEPD and show that this mutation renders DRG and trigeminal ganglion neurons hyperexcitable. These observations indicate that IEM and PEPD mutants are part of a physiological continuum that can produce a continuum of clinical phenotypes.

Phosphoribosylpyrophosphate synthesis in cultured human cells.

Phosphoribosylpyrophosphate (PRPP) concentrations and PRPP synthetase activity were studied in cultured human fibroblasts with control and mutant hypoxanthine-guanine phosphoribosylpyrophosphate (HPRT) activity. The PRPP concentrations increased 20- to 50-fold and PRPP synthetase activity 3-fold in cells from patients with the Lesch-Nyham syndrome when aminopterin, an inhibitor of de novo purine synthesis, was added to the medium. Concentrations of PRPP and PRPP synthetase activity did not increase in control cells in medium containing aminopterin unless hypoxanthine was removed from the medium. Exposure of cells to cycloheximide, a protein synthesis inhibitor, prevented the induction of PRPP synthetase and the formation of high PRPP concentrations. Cells from a patient with a mutant HPRT with a high Michaelis constant for PRPP increased PRPP levels and PRPP synthetase activity to a new steady state when they were grown in medium containing aminopterin. Inhibitors of de novo purine synthesis, the presence of hypoxanthine in the medium, and mutant HPRT activity affect the regulation of PRPP levels and PRPP synthetase activity.

Hypoxanthine-guanine phosphoribosyltransferase variant associated with accelerated purine synthesis.

We have previously described a 14-yr-old boy with hyperuricemia, renal failure, and accelerated purine production resistant in vivo and in vitro to purine analogs. This patient demonstrated normal red cell hypoxanthine-guanine phosphoribosyltransferase (HPRT) heat stability, electrophoresis at high pH, and activity at standard substrate levels. In the present report an abnormal HPRT enzyme was demonstrated by enzyme kinetic study with phosphoribosylpyrophosphate (PRPP) as the variable substrate and inhibitory studies with sodium fluoride. Apparently normal HPRT activity in a patient with hyperuricemia and gout does not exclude a functionally significant HPRT mutation.

Characterization of a neocentric supernumerary marker chromosome originating from the Xp distal region by FISH, CENP-C staining, and array CGH.

A small supernumerary marker chromosome (SMC) was observed in a girl with severe developmental delay. Her dysmorphism included prominent forehead, hypertelorism, down-slanting palpebral fissures, low-set/large ears, and flat nasal bridge with anteverted nares. This case also presented hypotonia, hypermobility of joints, congenital heart defect, umbilical hernia, failure to thrive, and seizures. The SMC originated from the distal region of Xp as identified by FISH with multiple DNA probes. Staining with antibodies to Centromere Protein C (CENP-C) demonstrated a neocentromere, while FISH with an alpha-satellite DNA probe showed no hybridization to the SMC. A karyotype was described as 47,XX,+neo(X)(pter-->p22.31::p22.31-->pter), indicating a partial tetrasomy of Xp22.31-->pter. This karyotype represents a functional trisomy for Xp22.31-->pter and a functional tetrasomy for the pseudoautosomal region given that there is no X-inactivation center in the marker chromosome. The SMC was further characterized by microarray-based comparative genomic hybridization (array CGH) as a duplicated DNA fragment of approximately 13 megabase pairs containing about 100 genes. We have described here a new neocentromere with discussion of its clinical significance.

FG syndrome update 1988: note of 5 new patients and bibliography.

At the eve of its mapping, the pre-molecular picture of the FG syndrome is heavily biased towards the severe end of the phenotypic spectrum because present knowledge is largely based on propositi. It is an X-linked, incompletely recessive, complexly pleiotropic syndrome with considerably variable expressivity. Though a true multiple congenital anomalies/mental retardation (MCA/MR) syndrome, severe malformations are uncommon and involve mostly the anus (60%) and non-colonic GI defects (33%), hypospadias (25%), cleft palate (6%), rarely a congenital heart defect. The complex CNS dysfunctions of congenital hypotonia and all of its sequelae, MR, and occasional seizures, must be attributed to a developmental CNS defect which is rarely demonstrated at pre-mortem, and which is known to involve agenesis of the corpus callosum in some 25% of appropriately studied patients (mostly propositi). Thus, the diagnosis is largely made on a specific constellation of minor anomalies and mild malformations in a hypotonic boy with severe constipation and a very characteristic facial appearance and behavioral phenotype. In about 1/3 of cases, carrier manifestations may be detected physically. New hemizygote manifestations seen in this review of 5 new patients include abnormal eruption of teeth, diastasis between upper central incisors, apparent gynecomastia, cleft lip, and nasolacrimal and helicine fistulae. Only a half hundred or so FG syndrome patients are known, but we suspect the syndrome is much more common than realized, and because of the unfortunate recurrence risk potential, deserves careful consideration in every appropriate case. RFLP mapping studies are urged in order to aid diagnosis of "mild" cases, and prenatal and carrier detection.

Congenital hypothalamic hamartoblastoma, hypopituitarism, imperforate anus and postaxial polydactyly--a new syndrome? Part I: clinical, causal, and pathogenetic considerations.

We report on six infants with a neonatally lethal malformation syndrome of hypothalamic hamartoblastoma, postaxial polydactyly, and imperforate anus. Some, but not all, patients had laryngeal cleft, abnormal lung lobulation, renal agenesis and/or renal dysplasia, short 4th metacarpals, nail dysplasia, multiple buccal frenula, hypoadrenalism, microphallus, congenital heart defect, and intrauterine growth retardation. The infants also had hypopituitarism and hypoadrenalism. All were sporadic cases, parents were not consanguineous, chromosomes were apparently normal. Family histories were unremarkable. There was insecticide and/or herbicide exposure in several of the cases, but no exposures were common to all 6 mothers. Five of the patients were born within an 8-month period, but all in different geographic locations. It is postulated that this is a previously apparently unreported syndrome of presently unknown cause.

Phytohemagglutinin-stimulated lymphocytes from patients with systemic lupus erythematosus demonstrate DNA damage.

DNA samples from control and lupus lymphocytes were studied for DNA integrity and single-strand breaks by agarose gel electrophoresis following digestion with the enzyme S1 nuclease. S1 nuclease digests single-strand gaps in double-stranded DNA. Gel patterns of phytohemagglutinin (PHA)-stimulated control and lupus lymphocyte DNAs were identical in the absence of S1 nuclease incubation. DNA isolated from PHA-stimulated control lymphocytes was relatively resistant to S1 nuclease digestion in 14 of 16 samples. However, 15 of 16 DNA samples from PHA-stimulated lupus lymphocytes demonstrated dramatically greater S1 nuclease digestion than paired control DNAs from lymphocytes analyzed at the same time under the same conditions. Increased S1 sensitivity suggests that more single-strand DNA breaks were found in PHA-stimulated lupus lymphocytes and/or the lupus DNA was more damaged than control DNA. We suggest that structural changes found in DNA from stimulated T lymphocytes of lupus patients are consistent with an endogenous antigen-mediated disorder.

Scavengers of free radical oxygen affect the generation of low molecular weight DNA in stimulated lymphocytes from patients with systemic lupus erythematosus.

Factors that potentially affect the generation of excess low molecular weight DNA (LMW-DNA) in cultured phytohemagglutinin (PHA)-stimulated lymphocytes of patients with systemic lupus erythematosus (SLE) were studied because this species of DNA is consistently found and this DNA may play a role in the pathogenesis of the disease. Superoxide dismutase (SOD; 0.05 mg/mL), a scavenger of free radical oxygen, decrease LMW-DNA formation in lymphocytes by 22%. Co-cultivation with cysteamine, a second scavenger of free radical oxygen and a sulfhydryl radioprotective agent, resulted in a 32% decrease in the generation of excess LMW-DNA at a concentration of 0.5 x 10(-3) mol/L and largely prevented its formation at 1.0 x 10(-3) mol/L. Other free radical scavengers (catalase, mannitol, vitamins C and E), cyclooxygenase inhibitors (ibuprofen and aspirin), a xanthine oxidase inhibitor (allopurinol), and an iron chelator (desferoxamine) did not affect excess LMW-DNA formation. Glutathione (1 x 10(-3) mol/L) had no effect and cysteine was toxic. Because scavengers of free radicals might be useful in the therapy of lupus, a trial of cysteamine (30 to 60 mg/kg/d) was administered to six acutely ill patients with SLE. A therapeutic benefit was not demonstrated, and some patients had exacerbation of disease. Lymphocyte cell growth from control and lupus subjects was stimulated when cysteamine, 1 x 10(-5) to 1 x 10(-4) mol/L was added to the media, but inhibited at concentrations of 2 x 10(-4) mol/L or greater. These studies suggest that the autooxidation and toxicity of high-dose cysteamine preclude its therapeutic use as a free radical scavenger.

Molecular, metabolic and immune evidence suggest that systemic autoimmune disease is antigen-mediated.

Patients with systemic lupus erythematosus generate a sustained immune response against self. The tools of modern molecular biology have been applied to cell activities and elements/signals of the immune system, but a structural or regulatory defect has not been found. When deoxyribonucleic acids for autoantibodies were cloned and sequenced, they were like other autoantibody DNA sequences; when genetic materials for autoantibodies were inserted into transgenic mice, cells secreting the antibodies were subject to normal control mechanisms and eliminated. A failure to clear self-reactive antibody producing thymocytes has not been demonstrated in human systemic lupus erythematosus. Molecular analyses of the efferent side of the immune response have been largely normal in systemic lupus erythematosus. The structure of autoantibodies suggests that they have been generated by selection pressures and the presence of endogenous antigens. If the immune system attack on self was secondary, structural changes and metabolic reactions capable of generating antigens should be found in systemic lupus erythematosus cells. Structural changes have been found in deoxyribonucleic acid from phytohaemagglutinin-stimulated systemic lupus erythematosus lymphocytes in the form of S1 nuclease-sensitive deoxyribonucleic acid breaks. Altered cellular macromolecules could result from endogenous metabolic processes, particularly oxygen free radicals and arachidonic acid metabolites. Excess free-radical species, generating positive nitroblue tetrazolium-reacting material and positive chemiluminescence, have been found in most but not all phytohaemagglutinin-stimulated lupus lymphocyte samples. If endogenous metabolic processes act on endogenous deoxyribonucleic acid, endogenous cell DNA breakdown may lead to low molecular weight deoxyribonucleic acids and deoxyribonucleic acid/immune complexes in systemic lupus erythematosus sera that are potentially immunogenic. These combined findings suggest that the exaggerated immune responses of systemic lupus erythematosus may be a normal response to protect the host from a perceived antigenic threat.

Characterization of an analphoid supernumerary marker chromosome derived from 15q25-->qter using high-resolution CGH and multiplex FISH analyses.

Supernumerary marker chromosomes (SMCs) without detectable alphoid DNA are predicted to have a neocentromere and have been referred to as mitotically stable neocentromere marker chromosomes (NMCs). We report the molecular cytogenetic characterization of a new case with analphoid NMC derived from 15q25-->qter using high-resolution comparative genomic hybridization (HR-CGH) and multiplex fluorescence in situ hybridization analyses with various alpha-satellite DNA probes, all-human-centromere probe (AHC), whole chromosome painting probes, and a subtelomere probe. The propositus is a dysmorphic infant who, at age 3 months, showed accelerated growth, partial deafness, and a phenotype similar to that of the eight previously reported cases of distal 15q tetrasomy. Chromosome studies showed that he had a de novo extra SMC in 80% of cells examined. HR-CGH revealed rev ish enh(15)(q25qter). Molecular cytogenetic analysis and molecular DNA polymorphism study demonstrated that this extra SMC is an NMC containing an inverted duplication of the distal long arm of chromosome 15 (tetrasomy 15q25-->qter) which originated paternally, i.e. ish der(15)(qte-->q25::q25[neocen]-->qter)(AHC-, CEP15-, WCP15+, PCP15q++). This case further elucidates the phenotype related to tetrasomy of this specific chromosome segment and represents a new report of a neocentromere on distal chromosome 15q suggesting that this region appears to be susceptible to the formation of neocentromeres.

The isotretinoin teratogen syndrome.

Two infants with prominent frontal bossing, hydrocephalus, microphthalmia, and small, malformed, low-set, undifferentiated ears were born to mothers who had taken isotretinoin in the first trimester of pregnancy. A Dandy-Walker malformation, microcephaly, hypertelorism, small ear canals, cleft palate, small mouth, and congenital heart disease were also observed. Isotretinoin is a potent teratogen in man; maternal ingestion early in pregnancy leads to a distinct clinical pattern of anomalies.

Recurrence of holoprosencephaly in families with a positive history.

A holoprosencephalic child was born into a family with 6 other affected members in 3 generations. A chromosome study of the proband was normal. The recurrence risk of holoprosencephaly was 22% in first degree relatives of affected individuals in this family. The recurrence risk was 22.9% for holoprosencephaly and 34.3% for holoprosencephaly and facial and neural defects when families with affected members in the medical literature in two generations were added. Dominant inheritance with reduced penetrance in family members best explains the inheritance pattern of familial holoprosencephaly.

Purine dysfunction in cells from patients with adenosine deaminase deficiency.

Conversion of adenosine to inosine is decreased in adenosine deaminase (ADA)-deficient fibroblasts at all concentrations of adenosine tested. Adenosine is not differentially toxic to ADA-deficient fibroblasts except at very high (5 X 10(-4) -1 X 10(-3) M) adenosine levels. Conversion of [14C] adenosine to GTP is not decreased in ADA-deficient cells compared with control cell strains. Adenosine conversion to ATP is the same as that in mutant cells except at high nonphysiologic concentrations, at which it is slightly decreased in ADA-deficient fibroblasts. This effect is probably not related to the biochemical pathology of ADA-deficient lymphocytes in vivo. Uridine, a pyrimidine compound, "rescues" control cells from the effects of adenosine toxicity, as previously reported, but it has no protective effect on ADA-deficient fibroblasts. This suggests that uridine will have no therapeutic role in the treatment of the ADA-deficient form of severe combined immunodeficiency (SCID) disease.

Epithelial cells and Von Gierke's disease.

Epithelial cells and not fibroblasts from human liver and amniotic fluid contain inducible glucose-6-phosphatase (G-6-Pase) activity. The diagnosis of Von Gierke's disease has been made in a patient with hepatomegaly utilizing cultured epithelial cells grown from a liver biopsy. G-6-Pase activity in epithelial cells from this patient could not be induced by dibutyryl cyclic AMP and theophylline. This is the first use of epithelial cells for diagnosis of a metabolic disease. G-6-Pase activity in cloned epithelial cells from amniotic fluid increases 2- to 3-fold after 24-hr exposure to dibutyryl cyclic AMP and theophylline. The prenatal diagnosis of Von Gierke's disease may be possible in a laboratory experienced with these techniques if epithelial cell growth is obtained from amniotic fluid.