Feline precision medicine using whole-exome sequencing identifies a novel frameshift mutation for vitamin D-dependent rickets type 2.

OBJECTIVES: A 14-week-old female domestic longhair kitten presented with shifting lameness and disproportionately smaller size compared with a co-housed littermate. METHODS: Hematology and serum biochemical testing were conducted to investigate causes for delayed growth, and radiographs of the appendicular skeleton were obtained. RESULTS: The afflicted kitten had marked hypocalcemia, mild hypophosphatemia and substantial elevations in alkaline phosphatase activity, as well as pathognomonic radiographic findings consistent with rickets. Skeletal changes and hypocalcemia prompted testing of concentrations of parathyroid hormone (PTH) and vitamin D metabolites. Endocrine testing demonstrated significant increases in serum concentrations of PTH and 1,25-dihydroxycholecalciferol (calcitriol), supporting a diagnosis of vitamin D-dependent rickets type 2. Provision of analgesia, supraphysiologic doses of calcitriol and calcium carbonate supplementation achieved normalization of the serum calcium concentration and restoration of normal growth, although some skeletal abnormalities persisted. Once skeletally mature, ongoing calcitriol supplementation was not required. Whole-exome sequencing (WES) was conducted to identify the underlying DNA variant. A cytosine deletion at cat chromosome position B4:76777621 in VDR (ENSFCAT00000029466:c.106delC) was identified and predicted to cause a stop codon in exon 2 (p.Arg36Glufs*18), disrupting >90% of the receptor. The variant was unique and homozygous in this patient and absent in the sibling and approximately 400 other cats for which whole-genome and whole-exome data were available. CONCLUSIONS AND RELEVANCE: A unique, heritable form of rickets was diagnosed in a domestic longhair cat. WES identified a novel frameshift mutation affecting the gene coding for the vitamin D3 receptor, determining the likely causal genetic variant. Precision medicine techniques, including whole-exome and whole-genome sequencing, can be a standard of care in cats to identify disease etiologies, and to target therapeutics and personalize treatment.

Biallelic frameshift variants in PHLDB1 cause mild-type osteogenesis imperfecta with regressive spondylometaphyseal changes.

BACKGROUND: Osteogenesis imperfecta (OI) is a heterogeneous group of inherited disorders characterised by susceptibility to fractures, primarily due to defects in type 1 collagen. The aim of this study is to present a novel OI phenotype and its causative candidate gene. METHODS: Whole-exome sequencing and clinical evaluation were performed in five patients from two unrelated families. PHLDB1 mRNA expression in blood and fibroblasts was investigated by real-time PCR, and western blot analysis was further performed on skin fibroblasts. RESULTS: The common findings among the five affected children were recurrent fractures and/or osteopaenia, platyspondyly, short and bowed long bones, and widened metaphyses. Metaphyseal and vertebral changes regressed after early childhood, and no fractures occurred under bisphosphonate treatment. We identified biallelic NM_001144758.3:c.2392dup and NM_001144758.3:c.2690_2693del pathogenic variants in PHLDB1 in the affected patients, respectively, in the families; parents were heterozygous for these variants. PHLDB1 encodes pleckstrin homology-like domain family B member-1 (PHLDB1) protein, which has a role in insulin-dependent Akt phosphorylation. Compared with controls, a decrease in the expression levels of PHLDB1 in the blood and skin fibroblast samples was detected. Western blot analysis of cultured fibroblasts further confirmed the loss of PHLDB1. CONCLUSION: Two biallelic frameshift variants in the candidate gene PHLDB1 were identified in independent families with a novel, mild-type, autosomal recessive OI. The demonstration of decreased PHLDB1 mRNA expression levels in blood and fibroblast samples supports the hypothesis that PHLDB1 pathogenic variants are causative for the observed phenotype.

A germline c.1546dupC MEN1 mutation in an MEN1 family: A case report.

RATIONALE: Multiple endocrine neoplasia type 1 (MEN1) is a rare tumor syndrome with an autosomal dominant inheritance, and genetic testing for MEN1 gene is important for both affected individuals and their relatives. We present a 2-person family affected by a germline c.1546dupC MEN1 mutation, and one of them had a full-spectrum of MEN-related endocrine tumors. PATIENT CONCERNS: A female patient aged 32 years presented with jejunal ulcer perforation due to gastrinoma. DIAGNOSES: We conducted genetic analysis and extensive biochemical/radiological evaluation for detecting other endocrine tumors. Multiple pancreatic neuroendocrine tumors (NETs), prolactinoma and primary hyperparathyroidism were diagnosed, and a frame-shift mutation, NM_130799.1:c.1546dupC (p.Arg516Profs∗15), was detected. One daughter of the proband, aged 12 years, had the same mutation for MEN1. INTERVENTION: She underwent pancreatic surgery for pancreatic NETs and total parathyroidectomy for primary hyperparathyroidism. OUTCOMES: After pancreatic surgery, long-term symptoms of epigastric soreness, acid belching, sweating, and palpitation in fasting were improved. Hypercalcemia was improved after parathyroidectomy and she was supplemented with oral calcium and vitamin D. Her daughter showed normal biochemical surveillance until 15 years of age. LESSONS: We report 2 people in a family affected by MEN1 with the heterozygous germline c.1546dupC mutation, a variant that should be surveilled for early development of full-blown MEN1-associated endocrine tumors.

A novel GNAS mutation in pseudohypoparathyroidism type 1a in a Chinese man presented with recurrent seizure: a case report.

BACKGROUND: Pseudohypoparathyroidism is a rare genetic disease characterized by hypocalcaemia and hyperphosphataemia due to the defect to the guanine nucleotide-binding protein alpha subunit (GNAS) gene. Patients with pseudoparathyroidism type 1a and 1c could manifest Albright's hereditary osteodystrophy and multiple hormone resistance including gonadotropin and thyroid stimulating hormone. CASE PRESENTATION: Here we report a Chinese man who presented with fatigue, recurrent seizure and Albright's hereditary osteodystrophy. His genetic study revealed a heterozygote mutation in the GNAS gene [NM_000516.4(GNAS): c2787_2788del (p.Val930AspfsTer12)]. After calcium and calcitriol supplement, his seizures achieved partially remission. CONCLUSIONS: We report a case of PHP1a or 1c with a novel frameshift mutation in GNAS gene in a patient presenting with AHO, as well as TSH and partial gonadotropin resistance. This mutation in this case has not been reported in literature and adds to the spectrum of genetic mutations related to PHP.

Type 1 Plasminogen Deficiency With Pulmonary Involvement: Novel Treatment and Novel Mutation.

Type 1 plasminogen deficiency is a rare genetic disorder. Type 1 plasminogen deficiency is characterized by fibrin-rich pseudomembrane formation on mucosal surfaces, particularly the conjunctiva. Tracheobronchial tree involvement is a less common reported manifestation of type 1 plasminogen deficiency. Pseudomembranes in the tracheobronchial tree may result in respiratory compromise and ultimately fail if not recognized and treated. Currently, there is no specific replacement therapy approved for the treatment of congenital plasminogen deficiency. In the present paper, we report that type 1 plasminogen deficiency with novel frameshift mutation and pulmonary involvement was treated initially with systemic fresh frozen plasma followed by pulmonary lavage with fresh frozen plasma and tissue plasminogen activator.

CRISPR-induced indels and base editing using the Staphylococcus aureus Cas9 in potato.

Genome editing is now widely used in plant science for both basic research and molecular crop breeding. The clustered regularly interspaced short palindromic repeats (CRISPR) technology, through its precision, high efficiency and versatility, allows for editing of many sites in plant genomes. This system has been highly successful to produce knock-out mutants through the introduction of frameshift mutations due to error-prone repair pathways. Nevertheless, recent new CRISPR-based technologies such as base editing and prime editing can generate precise and on demand nucleotide conversion, allowing for fine-tuning of protein function and generating gain-of-function mutants. However, genome editing through CRISPR systems still have some drawbacks and limitations, such as the PAM restriction and the need for more diversity in CRISPR tools to mediate different simultaneous catalytic activities. In this study, we successfully used the CRISPR-Cas9 system from Staphylococcus aureus (SaCas9) for the introduction of frameshift mutations in the tetraploid genome of the cultivated potato (Solanum tuberosum). We also developed a S. aureus-cytosine base editor that mediate nucleotide conversions, allowing for precise modification of specific residues or regulatory elements in potato. Our proof-of-concept in potato expand the plant dicot CRISPR toolbox for biotechnology and precision breeding applications.

Somatic Mutations and Intratumoral Heterogeneity of Cancer-Related Genes NLK, YY1 and PA2G4 in Gastric and Colorectal Cancers.

Many genes act as both tumor suppressor gene (TSG) and proto-oncogene depending on cellular context and cancer type. Nemo-like kinase (NLK) encoding a serine/threonine kinase, Yin Yang 1 (YY1) encoding a zinc-finger transcription factor and PA2G4 encoding an ErbB3 binding protein have both of these two opposing functions. In the present study, we analyzed NLK, YY1 and PA2G4 frameshift mutations in sporadic GC and CRC with high microsatellite instability (MSI-H). Also, regional intratumoral heterogeneity (ITH) of frameshift mutations of these genes was analyzed in CRCs. We found frameshift mutations of NLK, YY1 and PA2G4 in CRC and GC with MSI-H (17/132: 12.9%), but not in those with MSS (0/90). Two (12.5%), one (6.3%) and one (6.3%) CRC (s) of the 16 CRCs exhibited ITH of NLK, YY1 and PA2G4 mutations among the 4-7 regions, suggesting that ITH of the frameshift mutations might be frequent in the CRCs. These results suggest that frameshift mutations of NLK, YY1 and PA2G4 along with the ITH might contribute to MSI-H cancer pathogenesis.

Severe clinical manifestation of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency associated with two novel mutations: a case report.

BACKGROUND: Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS) deficiency is an autosomal recessive inborn error of metabolism, which will give rise to failure of ketogenesis in liver during illness or fasting. It is a very rare disease with only a few patients reported worldwide, most of which had a good prognosis after proper therapies. CASE PRESENTATION: We report a 9-month-old boy with mHS deficiency presenting with unusually severe and persistent acidosis after diarrhea and reduced oral food intake. The metabolic acidosis persisted even after supplementation with sugar and alkaline solution. Blood purification and assisted respiration alleviated symptoms, but a second onset induced by respiratory infection several days later led to multiple organ failure and death. Urine organic acid analysis during the acute episode revealed a complex pattern of ketogenic dicarboxylic and 3-hydroxydicarboxylic aciduria with prominent elevation of glutaric acid and adipic acid, which seem to be specific to mHS deficiency. Plasma acylcarnitine analysis revealed elevated 3-hydroxybutyrylcarnitine and acetylcarnitine. This is the first report of elevated 3-hydroxybutyrylcarnitine in mHS deficiency. Whole exome sequencing revealed a novel compound heterozygous mutation in HMGCS2 (c.100C > T and c.1465delA). CONCLUSION: This severe case suggests the need for patients with mHS deficiency to avoid recurrent illness because it can induce severe metabolic crisis, possibly leading to death. Such patients may also require special treatment, such as blood purification. Urine organic acid profile during the acute episode may give a hint to the disease.

A Carbonic Anhydrase Pseudogene Sensitizes Select Brucella Lineages to Low CO2 Tension.

Brucella spp. are intracellular pathogens that cause a disease known as brucellosis. Though the genus is highly monomorphic at the genetic level, species have animal host preferences and some defining physiologic characteristics. Of note is the requirement for CO2 supplementation to cultivate particular species, which confounded early efforts to isolate B. abortus from diseased cattle. Differences in the capacity of Brucella species to assimilate CO2 are determined by mutations in the carbonic anhydrase gene, bcaA Ancestral single-nucleotide insertions in bcaA have resulted in frameshifted pseudogenes in B. abortus and B. ovis lineages, which underlie their inability to grow under the low CO2 tension of a standard atmosphere. Incubation of wild-type B. ovis in air selects for mutations that "rescue" a functional bcaA reading frame, which enables growth under low CO2 and enhances the growth rate under high CO2 Accordingly, we show that heterologous expression of functional Escherichia coli carbonic anhydrases enables B. ovis growth in air. Growth of B. ovis is acutely sensitive to a reduction in CO2 tension, while frame-rescued B. ovis mutants are insensitive to CO2 shifts. B. ovis initiates a gene expression program upon CO2 downshift that resembles the stringent response and results in transcriptional activation of its type IV secretion system. Our study provides evidence that loss-of-function insertion mutations in bcaA sensitize the response of B. ovis and B. abortus to reduced CO2 tension relative to that of other Brucella lineages. CO2-dependent starvation and virulence gene expression programs in these species may influence persistence or transmission in natural hosts.IMPORTANCEBrucella spp. are highly related, but they exhibit differences in animal host preference that must be determined by genome sequence differences. B. ovis and the majority of B. abortus strains require high CO2 tension to be cultivated in vitro and harbor conserved insertional mutations in the carbonic anhydrase gene, bcaA, which underlie this trait. Mutants that grow in a standard atmosphere, first reported nearly a century ago, are easily selected in the laboratory. These mutants harbor varied indel polymorphisms in bcaA that restore its consensus reading frame and rescue its function. Loss of bcaA function has evolved independently in the B. ovis and B. abortus lineages and results in a dramatically increased sensitivity to CO2 limitation.

Heterozygous Truncating Variants in POMP Escape Nonsense-Mediated Decay and Cause a Unique Immune Dysregulatory Syndrome.

The proteasome processes proteins to facilitate immune recognition and host defense. When inherently defective, it can lead to aberrant immunity resulting in a dysregulated response that can cause autoimmunity and/or autoinflammation. Biallelic or digenic loss-of-function variants in some of the proteasome subunits have been described as causing a primary immunodeficiency disease that manifests as a severe dysregulatory syndrome: chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE). Proteasome maturation protein (POMP) is a chaperone for proteasome assembly and is critical for the incorporation of catalytic subunits into the proteasome. Here, we characterize and describe POMP-related autoinflammation and immune dysregulation disease (PRAID) discovered in two unrelated individuals with a unique constellation of early-onset combined immunodeficiency, inflammatory neutrophilic dermatosis, and autoimmunity. We also begin to delineate a complex genetic mechanism whereby de novo heterozygous frameshift variants in the penultimate exon of POMP escape nonsense-mediated mRNA decay (NMD) and result in a truncated protein that perturbs proteasome assembly by a dominant-negative mechanism. To our knowledge, this mechanism has not been reported in any primary immunodeficiencies, autoinflammatory syndromes, or autoimmune diseases. Here, we define a unique hypo- and hyper-immune phenotype and report an immune dysregulation syndrome caused by frameshift mutations that escape NMD.

Identification of ATIC as a Novel Target for Chemoradiosensitization.

PURPOSE: Mutations in the gene encoding 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final 2 steps of the purine de novo biosynthetic pathway, were identified in a subject referred for radiation sensitivity testing. Functional studies were performed to determine whether ATIC inhibition was radiosensitizing and, if so, to elucidate the mechanism of this effect and determine whether small molecule inhibitors of ATIC could act as effective radiosensitizing agents. METHODS AND MATERIALS: Both small interfering RNA knockdown and small molecule inhibitors were used to inactivate ATIC in cell culture. Clonogenic survival assays, the neutral comet assay, and γH2AX staining were used to assess the effects of ATIC inhibition or depletion on cellular DNA damage responses. RESULTS: Depletion of ATIC or inhibition of its transformylase activity significantly reduced the surviving fraction of cells in clonogenic survival assays in multiple cancer cell lines. In the absence of ionizing radiation exposure, ATIC knockdown or chemical inhibition activated cell cycle checkpoints, shifting cells to the more radiosensitive G2/M phase of the cell cycle, and depleted cellular adenosine triphosphate but did not result in detectable DNA damage. Cells in which ATIC was knocked down or inhibited and then treated with ionizing radiation displayed increased numbers of DNA double-strand breaks and a delay in the repair of those breaks relative to irradiated, but otherwise untreated, controls. Supplementation of culture media with exogenous adenosine triphosphate ameliorated the DNA repair phenotypes. CONCLUSIONS: These findings implicate ATIC as an effective, and previously unrecognized, target for chemoradiosensitization and, more broadly, suggest that purine levels in cells might have an underappreciated role in modulating the efficiency of DNA damage responses that could be exploited in radiosensitizing strategies.

Patient with multiple acyl-CoA dehydrogenation deficiency disease and FLAD1 mutations benefits from riboflavin therapy.

Multiple acyl-CoA dehydrogenation deficiency is genetically heterogenous metabolic disease with mutations in genes involved in electron transfer to the mitochondrial respiratory chain. Disease symptoms vary from severe neonatal form to late-onset presentation with metabolic acidosis, lethargy, vomiting, muscle pain and weakness. Riboflavin therapy has been shown to ameliorate diseases symptoms in some of these patients. Recently, mutations in FAD synthase have been described to cause multiple acyl-CoA dehydrogenation deficiency. We describe here the effect of riboflavin supplementation therapy in a previously reported adult patient with multiple acyl-CoA dehydrogenation deficiency having compound heterozygous gene variations in FLAD1 (MIM: 610595) encoding FAD synthase. We present thorough clinical history including laboratory investigations, muscle MRI, muscle biopsy and spiroergometric analyses comprising of a follow-up of 20 years. Our data suggest that patients with adult-onset multiple acyl-CoA dehydrogenation deficiency with FLAD1 gene mutations also benefit from long-term riboflavin therapy.

MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction.

Sexual reproduction in flowering plants involves double fertilization, the union of two sperm from pollen with two sex cells in the female embryo sac. Modern plant breeders increasingly seek to circumvent this process to produce doubled haploid individuals, which derive from the chromosome-doubled cells of the haploid gametophyte. Doubled haploid production fixes recombinant haploid genomes in inbred lines, shaving years off the breeding process. Costly, genotype-dependent tissue culture methods are used in many crops, while seed-based in vivo doubled haploid systems are rare in nature and difficult to manage in breeding programmes. The multi-billion-dollar maize hybrid seed business, however, is supported by industrial doubled haploid pipelines using intraspecific crosses to in vivo haploid inducer males derived from Stock 6, first reported in 1959 (ref. 5), followed by colchicine treatment. Despite decades of use, the mode of action remains controversial. Here we establish, through fine mapping, genome sequencing, genetic complementation, and gene editing, that haploid induction in maize (Zea mays) is triggered by a frame-shift mutation in MATRILINEAL (MTL), a pollen-specific phospholipase, and that novel edits in MTL lead to a 6.7% haploid induction rate (the percentage of haploid progeny versus total progeny). Wild-type MTL protein localizes exclusively to sperm cytoplasm, and pollen RNA-sequence profiling identifies a suite of pollen-specific genes overexpressed during haploid induction, some of which may mediate the formation of haploid seed. These findings highlight the importance of male gamete cytoplasmic components to reproductive success and male genome transmittance. Given the conservation of MTL in the cereals, this discovery may enable development of in vivo haploid induction systems to accelerate breeding in crop plants.

Genetic analysis of Tunisian families with Usher syndrome type 1: toward improving early molecular diagnosis.

PURPOSE: Usher syndrome accounts for about 50% of all hereditary deaf-blindness cases. The most severe form of this syndrome, Usher syndrome type I (USH1), is characterized by profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. Six USH1 genes have been identified, MYO7A, CDH23, PCDH15, USH1C, SANS, and CIB2, encoding myosin VIIA, cadherin-23, protocadherin-15, harmonin, scaffold protein containing ankyrin repeats and a sterile alpha motif (SAM) domain, and calcium- and integrin-binding member 2, respectively. METHODS: In the present study, we recruited four Tunisian families with a diagnosis of USH1, together with healthy unrelated controls. Affected members underwent detailed audiologic and ocular examinations. We used the North African Deafness (NADf) chip to search for known North African mutations associated with USH. Then, we selected microsatellite markers covering USH1 known loci to genotype the DNA samples. Finally, we performed DNA sequencing of three known USH1 genes: MYO7A, PCDH15, and USH1C. RESULTS: Four biallelic mutations, all single base changes, were found in the MYO7A, USH1C, and PCDH15 genes. These mutations consist of a previously reported splicing defect c.470+1G>A in MYO7A, three novel variants, including two nonsense (p.Arg3X and p.Arg134X) in USH1C and PCDH15, respectively, and one frameshift (p.Lys615Asnfs*6) in MYO7A. CONCLUSIONS: We found a remarkable genetic heterogeneity in the studied families with USH1 with a variety of mutations, among which three were novel. These novel mutations will be included in the NADf mutation screening chip that will allow a higher diagnosis efficiency of this extremely genetically heterogeneous disease. Ultimately, efficient molecular diagnosis of USH in a patient's early childhood is of utmost importance, allowing better educational and therapeutic management.

Long-term balancing selection at the Phosphorus Starvation Tolerance 1 (PSTOL1) locus in wild, domesticated and weedy rice (Oryza).

BACKGROUND: The ability to grow in phosphorus-depleted soils is an important trait for rice cultivation in many world regions, especially in the tropics. The Phosphorus Starvation Tolerance 1 (PSTOL1) gene has been identified as underlying the ability of some cultivated rice varieties to grow under low-phosphorus conditions; however, the gene is absent from other varieties. We assessed PSTOL1 presence/absence in a geographically diverse sample of wild, domesticated and weedy rice and sequenced the gene in samples where it is present. RESULTS: We find that the presence/absence polymorphism spans cultivated, weedy and wild Asian rice groups. For the subset of samples that carry PSTOL1, haplotype sequences suggest long-term selective maintenance of functional alleles, but with repeated evolution of loss-of-function alleles through premature stops and frameshift mutations. The loss-of-function alleles have evolved convergently in multiple rice species and cultivated rice varieties. Greenhouse assessments of plant growth under low- and high-phosphorus conditions did not reveal significant associations with PSTOL1 genotype variation; however, the striking signature of balancing selection at this locus suggests that further phenotypic characterizations of PSTOL1 allelic variants is warranted and may be useful for crop improvement. CONCLUSIONS: These findings suggest balancing selection for both functional and non-functional PSTOL1 alleles that predates and transcends Asian rice domestication, a pattern that may reflect fitness tradeoffs associated with geographical variation in soil phosphorus content.

Identification and Evolution of Functional Alleles of the Previously Described Pollen Specific Myrosinase Pseudogene AtTGG6 in Arabidopsis thaliana.

Myrosinases are ß-thioglucoside glucohydrolases and serve as defense mechanisms against insect pests and pathogens by producing toxic compounds. AtTGG6 in Arabidopsis thaliana was previously reported to be a myrosinase pseudogene but specifically expressed in pollen. However, we found that AlTGG6, an ortholog to AtTGG6 in A. lyrata (an outcrossing relative of A. thaliana) was functional, suggesting that functional AtTGG6 alleles may still exist in A. thaliana. AtTGG6 alleles in 29 A. thaliana ecotypes were cloned and sequenced. Results indicate that ten alleles were functional and encoded Myr II type myrosinase of 512 amino acids, and myrosinase activity was confirmed by overexpressing AtTGG6 in Pichia pastoris. However, the 19 other ecotypes had disabled alleles with highly polymorphic frame-shift mutations and diversified sequences. Thirteen frame-shift mutation types were identified, which occurred independently many times in the evolutionary history within a few thousand years. The functional allele was expressed specifically in pollen similar to the disabled alleles but at a higher expression level, suggesting its role in defense of pollen against insect pests such as pollen beetles. However, the defense function may have become less critical after A. thaliana evolved to self-fertilization, and thus resulted in loss of function in most ecotypes.

Assessment of antimutagenic action of Celtis glabrata Steven ex Planch. (Cannabaceae) extracts against base pair exchange and frame shift mutations on Salmonella typhimurium TA98 and TA100 strains by Ames test.

CONTEXT: Celtis glabrata is used in Turkey for the treatment of various health disorders. OBJECTIVE: The acetone, chloroform, ethanol, and methanol extracts of C. glabrata leaf, fruit, and seed were investigated to evaluate their antimutagenic activities. MATERIAL AND METHODS: The antimutagenicity of these extracts was determined by Ames test against mutagens (4-nitro-O-phenylenediamine, 2-aminofluorene (2-AF), and sodium azide (SA)). The extracts were used at concentrations between 5 and 0.005 mg/plate. RESULTS: The ethanol extracts of leaves exhibited strong antimutagenicity (70%) against 2-AF with S9 at 5 mg/plate on TA98. But methanol (61%, 53%) and acetone (53%, 52%) also revealed strong inhibition rates at concentrations of ≥ 0.5 mg/plate. Among the extracts, the highest activity (96%) was obtained from acetone extract against SA without S9, followed by chloroform extract (91%) at a dose of 5 mg/plate on TA100 with S9. Ethanol (without S9) and chloroform (with S9) extracts showed strong antimutagenicity at all doses. Exception of chloroform and acetone (without S9), all fruit extracts (with/without S9) manifested strong antimutagenicity at doses of ≥ 0.5 mg/plate on TA98 strain. Ethanol extracts revealed 68% inhibition against 2-AF on TA98. Acetone and ethanol extracts manifested 84% and 82% inhibition against SA on TA100, respectively. All the extracts of seeds revealed strong inhibition against 2-AF at ≥ 0.5 mg/plate doses on TA98, but acetone extract showed excellent antimutagenicity (94%). Moreover, the chloroform (74, 73, 63, 54%), acetone (74, 72, 70, 65%) and methanol (74, 67, 63, 61%) extracts of seeds revealed strong antimutagenic activity on TA100 against SA with S9. DISCUSSION AND CONCLUSION: This plant may be natural source of antimutagenic agents.

HDR syndrome in a Japanese girl with biliary atresia: a case report.

BACKGROUND: Hypoparathyroidism, sensorineural deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder. We report the first detailed case of hypoparathyroidism complicated by biliary atresia. CASE PRESENTATION: A 1-year-old Japanese girl was admitted to our hospital for living donor liver transplantation. She suffered from obstructive jaundice owing to biliary atresia. She also had persistent hypocalcemia. Despite oral calcium and abundant vitamin D supplementation, a laboratory test showed hypocalcemia (1.4 mmol/l) and hyperphosphatemia (2.6 mmol/l). The intact parathyroid hormone level was normal (66 ng/l) with severe vitamin D deficiency (25-hydroxy vitamin D: undetectable levels). There were no rachitic changes in metaphysis on X-rays. Her family history showed that her mother had sensorineural deafness, a low serum calcium level (2.1 mmol/l), hypoplastic left kidney, and a past history of an operation for right vesicoureteral reflux. We suspected that this patient and her mother have hypoparathyroidism, sensorineural deafness, and renal dysplasia syndrome. A heterozygous GATA3 gene mutation (c.736delGinsAT) was found in this patient and her mother, but not in her father. CONCLUSION: This familial case confirms the importance of family history in the diagnosis of HDR syndrome. Regardless of marked vitamin D deficiency, the complication of hypoparathyroidism prevented the onset of vitamin D deficiency rickets in our patient.

A case report of Gitelman syndrome resulting from two novel mutations in SLC12A3 gene.

INTRODUCTION: Hypokalaemia is a common clinical problem. A potential but commonly overlooked cause of hypokalaemia is Gitelman syndrome. MATERIAL AND METHODS: A 26-year-old man was admitted to the hospital due to syncope with general and muscular weakness and muscle cramps. The patient's history revealed previous recurrent syncope events associated to hypokalaemia with the lowest serum potassium value being 2.6mmol/l. At admission, blood pressure was normal and no changes were found at physical examination. Laboratory tests showed mild hypokalaemia (3.0mmol/l), hypomagnesaemia (1.36mg/dl), hypocalciuria (< 40mg/24h), and metabolic alkalosis (HCO3(-) 29.7mmol/l, BE 5.3mmol/l). RESULTS: Further laboratory tests (FeK, TTKG) confirmed inappropriate kaliuresis. Conn's disease was excluded by hormonal and imaging assessments. Genetic testing was performed and two novel heterozygous mutations: c.35_36insA and c.1095+5G>A were found in transcript NM_000339.2 in SLC12A3 gene. CONCLUSION: The patient was diagnosed with Gitelman syndrome and was treated with supplements of potassium and magnesium.