Carl Fratter

Identification of 70 calcium-sensing receptor mutations in hyper- and hypo-calcaemic patients: evidence for clustering of extracellular domain mutations at calcium-binding sites

The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that has an extracellular bilobed venus flytrap domain (VFTD) predicted to contain five calcium (Ca(2+))-binding sites. To elucidate the structure-function relationships of the VFTD, we investigated 294 unrelated probands with familial hypocalciuric hypercalcaemia (FHH), neonatal severe primary hyperparathyroidism (NSHPT) or autosomal dominant hypocalcaemic hypercalciuria (ADHH) for CaSR mutations and performed in vitro functional expression studies and three-dimensional modelling of mutations involving the VFTD. A total of 70 different CaSR mutations were identified: 35 in FHH, 10 in NSHPT and 25 in ADHH patients. Furthermore, a CaSR variant (Glu250Lys) was identified in FHH and ADHH probands and demonstrated to represent a functionally neutral polymorphism. NSHPT was associated with a large proportion of truncating CaSR mutations that occurred in the homozygous or compound heterozygous state. Thirty-four VFTD missense mutations were identified, and 18 mutations were located within 10 Å of one or more of the predicted Ca(2+)-binding sites, particularly at the VFTD cleft, which is the principal site of Ca(2+) binding. Mutations of residues 173 and 221, which are located at the entrance to the VFTD cleft binding site, were associated with both receptor activation (Leu173Phe and Pro221Leu) and inactivation (Leu173Pro and Pro221Gln), thereby highlighting the importance of these residues for entry and binding of Ca(2+) by the CaSR. Thus, these studies of disease-associated CaSR mutations have further elucidated the role of the VFTD cleft region in Ca(2+) binding and the function of the CaSR.

The clinical, histochemical, and molecular spectrum of PEO1 (Twinkle)-linked adPEO

Background: Mutations in the Twinkle (PEO1) gene are a recognized cause of autosomal dominant progressive external ophthalmoplegia (adPEO), resulting in the accumulation of multiple mitochondrial DNA (mtDNA) deletions and cytochrome c oxidase (COX)-deficient fibers in skeletal muscle secondary to a disorder of mtDNA maintenance. Patients typically present with isolated extraocular muscle involvement, with little apparent evidence of the clinical heterogeneity documented in other mtDNA maintenance disorders, in particular POLG-related disease. Methods: We reviewed the clinical, histochemical, and molecular genetics analysis of 33 unreported patients from 26 families together with all previous cases described in the literature to define the clinical phenotype associated with PEO1 mutations. Results: Ptosis and ophthalmoparesis were almost universal clinical features among this cohort, with 52% (17/33) reporting fatigue and 33% (11/33) having mild proximal myopathy. Features consistent with CNS involvement were rarely described; however, in 24% (8/33) of the patients, cardiac abnormalities were reported. Mitochondrial histochemical changes observed in muscle showed remarkable variability, as did the secondary mtDNA deletions, which in some patients were only detected by PCR-based assays and not Southern blotting. Moreover, we report 7 novel PEO1 variants. Conclusions: Our data suggest a shared clinical phenotype with variable mild multiorgan involvement, and that the contribution of PEO1 mutations as a cause of adPEO may well be underestimated. Direct sequencing of the PEO1 gene should be considered in adPEO patients prior to muscle biopsy.

Adults with RRM2B-related mitochondrial disease have distinct clinical and molecular characteristics.

Mutations in the nuclear-encoded mitochondrial maintenance gene RRM2B are an important cause of familial mitochondrial disease in both adults and children and represent the third most common cause of multiple mitochondrial DNA deletions in adults, following POLG [polymerase (DNA directed), gamma] and PEO1 (now called C10ORF2, encoding the Twinkle helicase) mutations. However, the clinico-pathological and molecular features of adults with RRM2B-related disease have not been clearly defined. In this multicentre study of 26 adult patients from 22 independent families, including five additional cases published in the literature, we show that extra-ocular neurological complications are common in adults with genetically confirmed RRM2B mutations. We also demonstrate a clear correlation between the clinical phenotype and the underlying genetic defect. Myopathy was a prominent manifestation, followed by bulbar dysfunction and fatigue. Sensorineural hearing loss and gastrointestinal disturbance were also important findings. Severe multisystem neurological disease was associated with recessively inherited compound heterozygous mutations with a mean age of disease onset at 7 years. Dominantly inherited heterozygous mutations were associated with a milder predominantly myopathic phenotype with a later mean age of disease onset at 46 years. Skeletal muscle biopsies revealed subsarcolemmal accumulation of mitochondria and/or cytochrome c oxidase-deficient fibres. Multiple mitochondrial DNA deletions were universally present in patients who underwent a muscle biopsy. We identified 18 different heterozygous RRM2B mutations within our cohort of patients, including five novel mutations that have not previously been reported. Despite marked clinical overlap between the mitochondrial maintenance genes, key clinical features such as bulbar dysfunction, hearing loss and gastrointestinal disturbance should help prioritize genetic testing towards RRM2B analysis, and sequencing of the gene may preclude performance of a muscle biopsy.

Collated mutations in mitochondrial DNA (mtDNA) depletion syndrome (excluding the mitochondrial gamma polymerase, POLG1).

These tables list both published and a number of unpublished mutations in genes associated with early onset defects in mitochondrial DNA (mtDNA) maintenance including C10orf2, SUCLG1, SUCLA2, TYMP, RRM2B, MPV17, DGUOK and TK2. The list should not be taken as evidence that any particular mutation is pathogenic. We have included genes known to cause mtDNA depletion, excluding POLG1, because of the existing database (http://tools.niehs.nih.gov/polg/). We have also excluded mutations in C10orf2 associated with dominant adult onset disorders.

RRM2B mutations are frequent in familial PEO with multiple mtDNA deletions

Disorders of mitochondrial DNA (mtDNA) maintenance leading to multiple mtDNA deletions are a significant cause of inherited neurologic disease in adults, but the underlying nuclear gene defects remain elusive in many patients. Following the recent description of a truncating mutation in the RRM2B gene—encoding the small subunit, p53R2, of the p53-inducible ribonucleotide reductase protein—in 2 families with autosomal-dominant progressive external ophthalmoplegia (adPEO),1 we determined the frequency of RRM2B mutations in a large cohort of patients with chronic PEO and multiple mtDNA deletions in muscle in whom mutations in all known candidate genes (e.g., POLG , POLG2 , SLC25A4 , and PEO1 ) had been excluded.2 ### Methods. We studied 75 unrelated probands with PEO, a mosaic defect of cytochrome c oxidase (COX) activity, and multiple mtDNA deletions in skeletal muscle who had been referred to Mitochondrial Diagnostic Centers at Newcastle, Oxford, or Munich for clinical assessment and histologic/molecular genetic analysis. The entire coding region, including intron–exon boundaries, of the RRM2B gene was determined as previously described.1 RRM2B exon copy number (exons 1–8) was assessed by MLPA (MRC-Holland kit P089-A1) in patients with single, heterozygous missense mutations. #### Standard protocol approvals, registrations, and patient consents. This study was approved and performed under the ethical guidelines issued by each institution for clinical studies, with written informed consent obtained from all subjects. ### Results. We identified 10 different …

Reversible infantile respiratory chain deficiency is a unique, genetically heterogenous mitochondrial disease

Objectives Homoplasmic maternally inherited, m.14674T>C or m. 14674T>G mt-tRNAGlu mutations have recently been identified in reversible infantile cytochrome c oxidase deficiency (or ‘benign COX deficiency’). This study sought other genetic defects that may give rise to similar presentations. Patients Eight patients from seven families with clinicopathological features of infantile reversible cytochrome c oxidase deficiency were investigated. Methods The study reviewed the diagnostic features and performed molecular genetic analyses of mitochondrial DNA and nuclear encoded candidate genes. Results Patients presented with subacute onset of profound hypotonia, feeding difficulties and lactic acidosis within the first months of life. Although recovery was remarkable, a mild myopathy persisted into adulthood. Histopathological findings in muscle included increased lipid and/or glycogen content, ragged-red and COX negative fibres. Biochemical studies suggested more generalised abnormalities than pure COX deficiency. Clinical improvement was reflected by normalisation of lactic acidosis and histopathological abnormalities. The m.14674T>C mt-tRNAGlu mutation was identified in four families, but none had the m. 14674T>G mutation. Furthermore, in two families pathogenic mutations were also found in the nuclear TRMU gene which has not previously been associated with this phenotype. In one family, the genetic aetiology still remains unknown. Conclusions Benign COX deficiency is better described as ‘reversible infantile respiratory chain deficiency’. It is genetically heterogeneous, and patients not carrying the m.14674T>C or T>G mt-tRNAGlu mutations may have mutations in the TRMU gene. Diagnosing this disorder at the molecular level is a significant advance for paediatric neurologists and intensive care paediatricians, enabling them to select children with an excellent prognosis for continuing respiratory support from those with severe mitochondrial presentation in infancy.

Clinical, biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are severe autosomal recessive disorders associated with decreased mtDNA copy number in clinically affected tissues. The hepatocerebral form (mtDNA depletion in liver and brain) has been associated with mutations in the POLG, PEO1 (Twinkle), DGUOK and MPV17 genes, the latter encoding a mitochondrial inner membrane protein of unknown function. The aims of this study were to clarify further the clinical, biochemical, cellular and molecular genetic features associated with MDS due to MPV17 gene mutations. We identified 12 pathogenic mutations in the MPV17 gene, of which 11 are novel, in 17 patients from 12 families. All patients manifested liver disease. Poor feeding, hypoglycaemia, raised serum lactate, hypotonia and faltering growth were common presenting features. mtDNA depletion in liver was demonstrated in all seven cases where liver tissue was available. Mosaic mtDNA depletion was found in primary fibroblasts by PicoGreen staining. These results confirm that MPV17 mutations are an important cause of hepatocerebral mtDNA depletion syndrome, and provide the first demonstration of mosaic mtDNA depletion in human MPV17 mutant fibroblast cultures. We found that a severe clinical phenotype was associated with profound tissue-specific mtDNA depletion in liver, and, in some cases, mosaic mtDNA depletion in fibroblasts.

A Missense Glial Cells Missing Homolog B (GCMB) Mutation, Asn502His, Causes Autosomal Dominant Hypoparathyroidism

CONTEXT Glial cells missing B (GCMB), the mammalian homolog of the Drosophila GCM gene, encodes a 506-amino-acid parathyroid-specific transcription factor. To date, only two different heterozygous GCMB mutations have been reported in three kindreds with autosomal dominant hypoparathyroidism. OBJECTIVE Our objective was to investigate a family with autosomal dominant hypoparathyroidism for PTH, CaSR, and GCMB mutations. METHODS Leukocyte DNA was used with exon-specific primers for PCR amplification and the DNA sequences of the PCR products determined. Functional analyses using fluorescence microscopy, EMSAs, and luciferase reporter assays were undertaken. Informed consent was obtained using protocols approved by a national ethical committee. RESULTS DNA sequence analysis revealed an A to C transversion at codon 502 of GCMB, which altered the wild-type asparagine (Asn) to histidine (His). Functional studies, using transient transfections of COS7 cells with GCMB wild-type and mutant (Asn502His) tagged constructs, demonstrated that the wild-type and mutant proteins localized to the nucleus and retained the ability to bind the GCM-consensus DNA recognition motif. However, a luciferase reporter assay demonstrated that the Asn502His mutation resulted in a reduction in gene transactivation. Moreover, cotransfection of the wild-type with mutant did not lead to an increase in luciferase activity, thereby demonstrating a dominant-negative effect of the Asn502His mutant that would be consistent with an autosomal dominant inheritance. CONCLUSION Our results, which have identified the first dominant missense GCMB mutation, help to increase our understanding of the mechanism underlying gene transactivation that is a prerequisite for the function of this parathyroid gland-specific transcription factor.

Information for genetic management of mtDNA disease: Sampling pathogenic mtDNA mutants in the human germline and in placenta.

Background Families with a child who died of severe, maternally inherited mitochondrial DNA (mtDNA) disease need information on recurrence risk. Estimating this risk is difficult because of (a) heteroplasmy—the coexistence of mutant and normal mtDNA in the same person—and (b) the so-called mitochondrial bottleneck, whereby the small number of mtDNAs that become the founders for the offspring cause variation in dose of mutant mtDNA. The timing of the bottleneck and of segregation of mtDNA during foetal life determines the management options. Therefore, mtDNA heteroplasmy was studied in oocytes and placenta of women in affected families. Results One mother of a child dying from Leigh syndrome due to the 9176T→C mtDNA mutation transmitted various loads of mutant mtDNA to ≤3 of 20 oocytes. This was used to estimate recurrence as ≤5%. She subsequently conceived a healthy son naturally. Analysis of the placenta showed that some segregation also occurred during placental development, with the mutant mtDNA load varying by >10% in a placenta carrying 65% 3243A→G mutant mtDNA. Discussion This is the first report of (a) an oocyte analysis for preconception counselling, specifically, refining recurrence risks of rare mutations and (b) a widely different load of a pathogenic mtDNA mutation in multiple oocytes, apparently confined to the germline, in an asymptomatic carrier of an mtDNA disease. This suggests that a major component of the bottleneck occurs during oogenesis, probably early in the foetal life of the mother. The variable mutant load in placenta implies that estimates based on a single sample in prenatal diagnosis of mtDNA disorders have limited accuracy.

Prospective study of POLG mutations presenting in children with intractable epilepsy: Prevalence and clinical features

To assess the frequency and clinical features of childhood‐onset intractable epilepsy caused by the most common mutations in the POLG gene, which encodes the catalytic subunit of mitochondrial DNA polymerase gamma.