A missense mutation in the skeletal muscle chloride channel 1 (CLCN1) as candidate causal mutation for congenital myotonia in a New Forest pony

A 7-month-old New Forest foal presented for episodes of recumbency and stiffness with myotonic discharges on electromyography. The observed phenotype resembled congenital myotonia caused by CLCN1 mutations in goats and humans. Mutation of the CLCN1 gene was considered as possible cause and mutation analysis was performed. The affected foal was homozygous for a missense mutation (c.1775A>C, p.D592A) located in a well conserved domain of the CLCN1 gene. The mutation showed a recessive mode of inheritance within the reported pony family. Therefore, this CLCN1 polymorphism is considered to be a possible cause of congenital myotonia.

Microphthalmia in Texel sheep is associated with a missense mutation in the paired-like homeodomain 3 (PITX3) gene

Microphthalmia in sheep is an autosomal recessive inherited congenital anomaly found within the Texel breed. It is characterized by extremely small or absent eyes and affected lambs are absolutely blind. For the first time, we use a genome-wide ovine SNP array for positional cloning of a Mendelian trait in sheep. Genotyping 23 cases and 23 controls using Illumina's OvineSNP50 BeadChip allowed us to localize the causative mutation for microphthalmia to a 2.4 Mb interval on sheep chromosome 22 by association and homozygosity mapping. The PITX3 gene is located within this interval and encodes a homeodomain-containing transcription factor involved in vertebrate lens formation. An abnormal development of the lens vesicle was shown to be the primary event in ovine microphthalmia. Therefore, we considered PITX3 a positional and functional candidate gene. An ovine BAC clone was sequenced, and after full-length cDNA cloning the PITX3 gene was annotated. Here we show that the ovine microphthalmia phenotype is perfectly associated with a missense mutation (c.338G>C, p.R113P) in the evolutionary conserved homeodomain of PITX3. Selection against this candidate causative mutation can now be used to eliminate microphthalmia from Texel sheep in production systems. Furthermore, the identification of a naturally occurring PITX3 mutation offers the opportunity to use the Texel as a genetically characterized large animal model for human microphthalmia.

Haematological parameters are normal in dominant white Franches-Montagnes horses carrying a KIT mutation

The KIT receptor protein-tyrosine kinase plays an important role during embryonic development. Activation of KIT is crucial for the development of various cell lineages such as melanoblasts, stem cells of the haematopoietic system, spermatogonia and intestinal cells of Cajal. In mice, many mutations in the Kit gene cause pigmentation disorders accompanied by pleiotropic effects on blood cells and male fertility. Previous work has demonstrated that dominant white Franches-Montagnes horses carry one copy of the KIT gene with the p.Y717X mutation. The targeted breeding of white horses would be ethically questionable if white horses were known to suffer from anaemia or leukopenia. The present study demonstrates that no statistically significant differences in peripheral blood parameters are detectable between dominant white and solid-coloured Franches-Montagnes horses. The data indicate that KIT mutations may have different effects in mice, pigs, and horses. The KIT p.Y717X mutation does not have a major negative effect on the haematopoietic system of dominant white horses.

Identification of the bovine Arachnomelia mutation by massively parallel sequencing implicates sulfite oxidase (SUOX) in bone development

Arachnomelia is a monogenic recessive defect of skeletal development in cattle. The causative mutation was previously mapped to a approximately 7 Mb interval on chromosome 5. Here we show that array-based sequence capture and massively parallel sequencing technology, combined with the typical family structure in livestock populations, facilitates the identification of the causative mutation. We re-sequenced the entire critical interval in a healthy partially inbred cow carrying one copy of the critical chromosome segment in its ancestral state and one copy of the same segment with the arachnomelia mutation, and we detected a single heterozygous position. The genetic makeup of several partially inbred cattle provides extremely strong support for the causality of this mutation. The mutation represents a single base insertion leading to a premature stop codon in the coding sequence of the SUOX gene and is perfectly associated with the arachnomelia phenotype. Our findings suggest an important role for sulfite oxidase in bone development.

The mutation causing the black-and-tan pigmentation phenotype of Mangalitza pigs maps to the porcine ASIP locus but does not affect its coding sequence

The gene for agouti signaling protein (ASIP) is centrally involved in the expression of coat color traits in animals. The Mangalitza pig breed is characterized by a black-and-tan phenotype with black dorsal pigmentation and yellow or white ventral pigmentation. We investigated a Mangalitza x Piétrain cross and observed a coat color segregation pattern in the F2 generation that can be explained by virtue of two alleles at the MC1R locus and two alleles at the ASIP locus. Complete linkage of the black-and-tan phenotype to microsatellite alleles at the ASIP locus on SSC 17q21 was observed. Corroborated by the knowledge of similar mouse coat color mutants, it seems therefore conceivable that the black-and-tan pigmentation of Mangalitza pigs is caused by an ASIP allele a(t), which is recessive to the wild-type allele A. Toward positional cloning of the a(t) mutation, a 200-kb genomic BAC/PAC contig of this chromosomal region has been constructed and subsequently sequenced. Full-length ASIP cDNAs obtained by RACE differed in their 5' untranslated regions, whereas they shared a common open reading frame. Comparative sequencing of all ASIP exons and ASIP cDNAs between Mangalitza and Piétrain pigs did not reveal any differences associated with the coat color phenotype. Relative qRT-PCR analyses showed different dorsoventral skin expression intensities of the five ASIP transcripts in black-and-tan Mangalitza. The a(t) mutation is therefore probably a regulatory ASIP mutation that alters its dorsoventral expression pattern.

A novel SCN5A mutation, F1344S, identified in a patient with Brugada syndrome and fever-induced ventricular fibrillation

OBJECTIVE: Brugada syndrome (BS) is an inherited electrical cardiac disorder characterized by right bundle branch block pattern and ST segment elevation in leads V1 to V3 on surface electrocardiogram that can potentially lead to malignant ventricular tachycardia and sudden cardiac death. About 20% of patients have mutations in the only so far identified gene, SCN5A, which encodes the alpha-subunit of the human cardiac voltage-dependent sodium channel (hNa(v)1.5). Fever has been shown to unmask or trigger the BS phenotype, but the associated molecular and the biophysical mechanisms are still poorly understood. We report on the identification and biophysical characterization of a novel heterozygous missense mutation in SCN5A, F1344S, in a 42-year-old male patient showing the BS phenotype leading to ventricular fibrillation during fever. METHODS: The mutation was reproduced in vitro using site-directed mutagenesis and characterized using the patch clamp technique in the whole-cell configuration. RESULTS: The biophysical characterization of the channels carrying the F1344S mutation revealed a 10 mV mid-point shift of the G/V curve toward more positive voltages during activation. Raising the temperature to 40.5 degrees C further shifted the mid-point activation by 18 mV and significantly changed the slope factor in Na(v)1.5/F1344S mutant channels from -6.49 to -10.27 mV. CONCLUSIONS: Our findings indicate for the first time that the shift in activation and change in the slope factor at a higher temperature mimicking fever could reduce sodium currents' amplitude and trigger the manifestation of the BS phenotype.

Clinical and biochemical description of a novel CYP21A2 gene mutation 962_963insA using a new 3D model for the P450c21 protein

OBJECTIVE: A severely virilized 46, XX newborn girl was referred to our center for evaluation and treatment of congenital adrenal hyperplasia (CAH) because of highly elevated 17alpha-hydroxyprogesterone levels at newborn screening; biochemical tests confirmed the diagnosis of salt-wasting CAH. Genetic analysis revealed that the girl was compound heterozygote for a previously reported Q318X mutation in exon 8 and a novel insertion of an adenine between nucleotides 962 and 963 in exon 4 of the CYP21A2 gene. This 962_963insA mutation created a frameshift leading to a stop codon at amino acid 161 of the P450c21 protein. AIM AND METHODS: To better understand structure-function relationships of mutant P450c21 proteins, we performed multiple sequence alignments of P450c21 with three mammalian P450s (P450 2C8, 2C9 and 2B4) with known structures as well as with human P450c17. Comparative molecular modeling of human P450c21 was then performed by MODELLER using the X-ray crystal structure of rabbit P450 2B4 as a template. RESULTS: The new three dimensional model of human P450c21 and the sequence alignment were found to be helpful in predicting the role of various amino acids in P450c21, especially those involved in heme binding and interaction with P450 oxidoreductase, the obligate electron donor. CONCLUSION: Our model will help in analyzing the genotype-phenotype relationship of P450c21 mutations which have not been tested for their functional activity in an in vitro assay.

A novel mutation in the steroidogenic acute regulatory protein gene promoter leading to reduced promoter activity

We have identified a novel cytosine/thymidine polymorphism of the human steroidogenic acute regulatory (StAR) gene promoter located 3 bp downstream of the steroidogenic factor-1 (SF-1)-binding site and 9 bp upstream of the TATA box (ATTTAAG). Carriers of this mutation have a high prevalence of primary aldosteronism. In transfection experiments, basal StAR promoter activity was unaltered by the mutation in murine Y-1 cells and human H295R cells. In Y-1 cells, forskolin (25 microM, 6 h) significantly increased wild-type promoter activity to 230+/-33% (P<0.05, n=4). In contrast, forskolin increased mutated promoter activity only to 150+/-27%, with a significant 35% reduction compared to wild type (P<0.05, n=3). In H295R cells, angiotensin II (AngII; 10 nM) increased wild-type StAR promoter activity to 265+/-22% (P<0.01, n=3), while mutated StAR promoter activity in response to AngII only reached 180+/-29% of controls (P< 0.01, n=3). Gel mobility shift assays show the formation of two additional complexes with the mutated promoter: one with the transcription repressor DAX-1 and another with a yet unidentified factor, which strongly binds the SF-1 response element. Thus, this novel mutation in the human StAR promoter is critically involved in the regulation of StAR gene expression and is associated with reduced promoter activity, a finding relevant for adrenal steroid response to physiological stimulators.

Paracellin-1 gene mutation with multiple congenital abnormalities

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is an autosomal recessive renal tubular disorder characterized by renal magnesium wasting, hypercalciuria, advanced nephrocalcinosis and progressive renal failure. Mutations in the paracellin-1 (CLDN16) gene have been defined as the underlying genetic defect. The tubular disorders and progression in renal failure are usually resistant to magnesium substitution and hydrochlorothiazide therapy, but hypomagnesemia may improve with advanced renal insufficiency. We present a patient with a homozygous truncating CLDN16 gene mutation (W237X) who had early onset of renal insufficiency despite early diagnosis at 2 months. He also had additional abnormalities including horseshoe kidney, neonatal teeth, atypical face, cardiac abnormalities including coarctation of the aorta associated with atrial and ventricular septal defects, umbilical hernia and hypertrichosis. To the best of our knowledge, this is the youngest case diagnosed as familial hypomagnesemia with hypercalciuria and nephrocalcinosis and the first case having such additional congenital abnormalities independent of the disease itself.

An unusual patient with hypercalciuria, recurrent nephrolithiasis, hypomagnesemia and G227R mutation of Paracellin-1. An unusual patient with hypercalciuria and hypomagnesemia unresponsive to thiazide diuretics

A 19-year-old female patient with hypercalciuria and recurrent nephrolithiasis/urinary tract infection unresponsive to thiazide type diuretics is presented. The patient first experienced nephrolithiasis at the age of 4 years. Afterwards, recurrent passages of stones and urinary tract infection occurred. On diagnostic evaluation at the age of 19 years, she also had hypocitraturia and hypomagnesemia. Her serum calcium concentrations were near the lower limit of normal (8.5-8.8 mg/dl; normal range: 8.5-10.5), her serum magnesium concentrations were 1.15-1.24 mg/dl (normal range: 1.4-2.5) and urinary calcium excretion was 900 mg/24 h. PTH concentrations were increased (110-156 pg/ml; normal range: 10-65). We tried to treat the patient with hydrochlorothiazide at a dose of 50 mg/day. During treatment with thiazide diuretics, PTH concentration remained high and the patient had recurrent urinary tract infections and passages of stones. Serum magnesium concentration did not normalize even under the parenteral magnesium infusion. Her mother had a history of nephrolithiasis 20 years ago. Severe hypomagnesemia in association with hypercalciuria/urinary stones is reported as a rare autosomal recessive disorder caused by impaired reabsorption of magnesium and calcium in the thick assending limp of Henle's loop. Recent studies showed that mutations in the CLDN16 gene encoding paracellin-1 cause the disorder. In exon 4, a homozygous nucleotide exchange (G679C) was identified for the patient. This results in a point mutation at position Glycine227, which is replaced by an Arginine residue (G227R). The mother was heterozygous for this mutation. G227 is located in the fourth transmembrane domain and is highly conserved in the claudin gene family. This case indicates the pathogenetic role of paracellin-1 mutation in familial hypomagnesemia with hypercalciuria and nephrocalcinosis and further underlines the risk of stone formation in heterozygous mutation carriers.

Hydrochlorothiazide in CLDN16 mutation

BACKGROUND: Hydrochlorothiazide (HCT) is applied in the therapy of familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC) caused by claudin-16 (CLDN16) mutation. However, the short-term efficacy of HCT to reduce hypercalciuria in FHHNC has not yet been demonstrated in a clinical trial. METHODS: Four male and four female patients with FHHNC and CLDN16 mutation, under long-standing HCT therapy (0.4-1.2 mg/kg, median 0.9 mg/kg, dose according to calciuria), aged 0.7-22.4 years, were included in a clinical study to investigate the effect of HCT on calciuria. The study design consisted of three periods: continued therapy for 4 weeks, HCT withdrawal for 6 weeks and restart of therapy at the same dose for 4 weeks. Calciuria and magnesiuria were assessed weekly as Ca/creat and Mg/creat ratio, every 2 weeks in 24 h urine, and serum Mg, K and kaliuria (s-Mg, s-K and K/creat) at weeks 0, 6, 10 and 14. The data of each study period were averaged and analysed by Friedman and Wilcoxon test. RESULTS: Ca/creat was significantly reduced by HCT (median before/at/after withdrawal 0.76/1.24/0.77 mol/mol creat; n = 8, P<0.05). The reduction of Ca/24 h by HCT was not statistically significant (0.13/0.19/0.13 mmol/kg x 24 h; n = 5). Serum Mg (0.51/0.64/0.56 mmol/l; n = 8, P<0.05) and Serum K (3.65/4.35/3.65 mmol/l; n = 8, P<0.05) were significantly higher during withdrawal. However, Mg/creat (0.98/0.90/0.90 mol/mol creat; n = 8), Mg/24 h (0.14/0.12/0.18 mmol/kg x 24h; n = 5) and K/creat (6.3/8.4/6.2 mol/mol creat; n = 8) remained statistically unchanged during withdrawal. CONCLUSIONS: We demonstrated that HCT is effective in reducing hypercalciuria due to CLDN16 mutation on a short-term basis. However, the efficacy of HCT to attenuate disease progression remains to be elucidated.

A Spanish founder mutation in the chloride channel gene, CLCNKB, as a cause of atypical Bartter syndrome in adult age

BACKGROUND: Mutations in the chloride channel gene, CLCNKB, usually cause classic Bartter syndrome (cBS) or a mixed Bartter-Gitelman phenotype in the first years of life. METHODS: We report an adult woman with atypical BS caused by a homozygous missense mutation, A204T, in the CLCNKB gene, which has previously been described as the apparently unique cause of cBS in Spain. RESULTS: The evaluation of this patient revealed an overlap of phenotypic features ranging from severe biochemical and systemic disturbances typical of cBS to scarce symptoms and diagnosis in the adult age typical of Gitelman syndrome. The tubular disease caused a dramatic effect on mental, growth and puberal development leading to low IQ, final short stature and abnormal ovarian function. Furthermore, low serum PTH concentrations with concomitant nephrocalcinosis and normocalcaemia were observed. Both ovarian function and serum PTH levels were normalized after treatment with cyclooxygenase inhibitors. CONCLUSIONS: The present report confirms a weak genotype-phenotype correlation in patients with CLCNKB mutations and supports the founder effect of the A204T mutation in Spain. In our country, the genetic diagnosis of adult patients with hereditary hypokalaemic tubulopathies should include a screening of A204T mutation in the CLCNKB gene.