Novel CAPN3 variant associated with an autosomal dominant calpainopathy.

AIMS: The most common autosomal recessive limb girdle muscular dystrophy is associated with the CAPN3 gene. The exclusively recessive inheritance of this disorder has been recently challenged by the description of the recurrent variants, c.643_663del21 [p.(Ser215_Gly221del)] and c.598_612del15 [p.(Phe200_Leu204del)], associated with autosomal dominant inheritance. Our objective was to confirm the existence of autosomal dominant calpainopathies. METHODS: Through our activity as one of the reference centres for genetic diagnosis of calpainopathies in France and the resulting collaborations through the French National Network for Rare Neuromuscular Diseases (FILNEMUS), we identified four families harbouring the same CAPN3 heterozygous variant with supposedly autosomal dominant inheritance. RESULTS: We identified a novel dominantly inherited CAPN3 variant, c.1333G>A [p.(Gly445Arg)] in 14 affected patients from four unrelated families. The complementary phenotypic, functional and genetic findings correlate with an autosomal dominant inheritance in these families, emphasizing the existence of this novel transmission mode for calpainopathies. The mild phenotype associated with these autosomal dominant cases widens the phenotypic spectrum of calpainopathies and should therefore be considered in clinical practice. CONCLUSIONS: We confirm the existence of autosomal dominant calpainopathies as an entity beyond the cases related to the in-frame deletions c.643_663del21 and c.598_612del15, with the identification of a novel dominantly inherited and well-documented CAPN3 missense variant, c.1333G>A [p.(Gly445Arg)]. In addition to the consequences for genetic counselling, the confirmation of an autosomal dominant transmission mode for calpainopathies underlines the importance of re-assessing other myopathies for which the inheritance is considered as strictly autosomal recessive.

Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits.

Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is characterized by degeneration of the large sensory neurons and spinocerebellar tracts, cardiomyopathy and increased incidence in diabetes. FRDA is caused by severely reduced levels of frataxin, a mitochondrial protein of unknown function. Yeast knockout models as well as histological and biochemical data from heart biopsies or autopsies of FRDA patients have shown that frataxin defects cause a specific iron-sulfur protein deficiency and intramitochondrial iron accumulation. We have recently shown that complete absence of frataxin in the mouse leads to early embryonic lethality, demonstrating an important role for frataxin during mouse development. Through a conditional gene-targeting approach, we have generated in parallel a striated muscle frataxin-deficient line and a neuron/cardiac muscle frataxin-deficient line, which together reproduce important progressive pathophysiological and biochemical features of the human disease: cardiac hypertrophy without skeletal muscle involvement, large sensory neuron dysfunction without alteration of the small sensory and motor neurons, and deficient activities of complexes I-III of the respiratory chain and of the aconitases. Our models demonstrate time-dependent intramitochondrial iron accumulation in a frataxin-deficient mammal, which occurs after onset of the pathology and after inactivation of the Fe-S-dependent enzymes. These mutant mice represent the first mammalian models to evaluate treatment strategies for the human disease.

Homozygous SMN1 exons 1-6 deletion: pitfalls in genetic counseling and general recommendations for spinal muscular atrophy molecular diagnosis.

We report on a rare homozygous intragenic deletion encompassing exons 1-6 of the SMN1 gene in a patient with spinal muscular atrophy (SMA) born into a consanguineous family. This exceptional configuration induced misinterpretation of the molecular defect involved in this patient, who was first reported as having a classic SMN1 exon 7 deletion. This case points out the possible pitfalls in molecular diagnosis of SMA in affected patients and their relatives: exploration of the SMN1 exon 7 (c.840C/T alleles) may be disturbed by several non-pathological or pathological variants around the SMN1 exon 7. In order to accurately describe the molecular defect in an SMA-affected patient, we propose to apply the Human Genome Variation Society nomenclature. This widely accepted nomenclature would improve the reporting of the molecular defect observed in SMA patients and thus would avoid the commonly used but imprecise terminology "absence of SMN1 exon 7."

Miyoshi-like distal myopathy with mutations in anoctamin 5 gene.

Miyoshi myopathy is the most common form of recessive distal myopathy. Recessive mutations in the ANO5 gene have been recently identified in Northern Europe as a cause of non dysferlin-linked distal myopathy and limb girdle muscular dystrophy. We report here the first French cases of anoctamin 5 myopathy in 2 brothers presenting with a Miyoshi-like pattern. Comparing these patients with 12 other cases from the literature shows that all cases share a homogeneous clinical pattern, characterized by initial calf muscles involvement. Asymmetric muscle atrophy often precedes weakness. In this setting, high CK level and normal expression of dysferlin in muscle should lead to consider the diagnosis, which will be confirmed by ANO5 gene testing. The c.191dupA mutation, already reported as a founder mutation in Caucasian patients with anoctamin myopathies, was found in our family in a heterozygous state.

Noninvasive prenatal diagnosis of genetic diseases induced by triplet repeat expansion by linked read haplotyping and Bayesian approach.

The field of noninvasive prenatal diagnosis (NIPD) has undergone significant progress over the last decade. Direct haplotyping has been successfully applied for NIPD of few single-gene disorders. However, technical issues remain for triplet-repeat expansions. The objective of this study was to develop an NIPD approach for couples at risk of transmitting dynamic mutations. This method includes targeted enrichment for linked-read libraries and targeted maternal plasma DNA sequencing. We also developed an innovative Bayesian procedure to integrate the Hoobari fetal genotyping model for inferring the fetal haplotype and the targeted gene variant status. Our method of directly resolving parental haplotypes through targeted linked-read sequencing was smoothly performed using blood samples from families with Huntington's disease or myotonic dystrophy type 1. The Bayesian analysis of transmission of parental haplotypes allowed defining the genotype of five fetuses. The predicted variant status of four of these fetuses was in agreement with the invasive prenatal diagnosis findings. Conversely, no conclusive result was obtained for the NIPD of fragile X syndrome. Although improvements should be made to achieve clinically acceptable accuracy, our study shows that linked-read sequencing and parental haplotype phasing can be successfully used for NIPD of triplet-repeat expansion diseases.Trial registration: NCT04698551_date of first registration: 07/01/2021.

Identification of 28 novel mutations in the Bardet-Biedl syndrome genes: the burden of private mutations in an extensively heterogeneous disease.

Bardet-Biedl syndrome (BBS), an emblematic disease in the rapidly evolving field of ciliopathies, is characterized by pleiotropic clinical features and extensive genetic heterogeneity. To date, 14 BBS genes have been identified, 3 of which have been found mutated only in a single BBS family each (BBS11/TRIM32, BBS13/MKS1 and BBS14/MKS4/NPHP6). Previous reports of systematic mutation detection in large cohorts of BBS families (n > 90) have dealt only with a single gene, or at most small subsets of the known BBS genes. Here we report extensive analysis of a cohort of 174 BBS families for 12/14 genes, leading to the identification of 28 novel mutations. Two pathogenic mutations in a single gene have been found in 117 families, and a single heterozygous mutation in 17 families (of which 8 involve the BBS1 recurrent mutation, M390R). We confirm that BBS1 and BBS10 are the most frequently mutated genes, followed by BBS12. No mutations have been found in BBS11/TRIM32, the identification of which as a BBS gene only relies on a single missense mutation in a single consanguineous family. While a third variant allele has been observed in a few families, they are in most cases missenses of uncertain pathogenicity, contrasting with the type of mutations observed as two alleles in a single gene. We discuss the various strategies for diagnostic mutation detection, including homozygosity mapping and targeted arrays for the detection of previously reported mutations.

Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.

Friedreich's ataxia (FRDA) is an autosomal recessive, degenerative disease that involves the central and peripheral nervous systems and the heart. A gene, X25, was identified in the critical region for the FRDA locus on chromosome 9q13. This gene encodes a 210-amino acid protein, frataxin, that has homologs in distant species such as Caenorhabditis elegans and yeast. A few FRDA patients were found to have point mutations in X25, but the majority were homozygous for an unstable GAA trinucleotide expansion in the first X25 intron.

[Unilateral presentation of X-linked myotubular myopathy (XLMTM) in two out of three female carriers in a family with no affected male]. / Expression hémicorporelle d'une myopathie myotubulaire liée à l'X (XLMTM) chez deux des trois femmes conductrices d'une même famille sans cas masculin.

INTRODUCTION: X-linked myotubular myopathy (XLMTM), a recessive disorder, is caused by mutations affecting the myotubulatin (MTM1) gene located on the X chromosome. Most of the affected males die in the early postnatal period whereas female carriers are usually asymptomatic. CASE REPORTS: We report a family in which two females (45 and 27 years old) in two different generations, presented unilateral weakness which had worsened since adolescence, and one 48-year-old woman presented minimal symptoms. In agreement with the computed tomography and magnetic resonance imaging findings, the EMG was compatible with myopathy. Serum creatine kinase was elevated in the second patient. The histological study showed centronuclear myopathy aspects, more severe in the second patient. Both presented c.1420C>T, p.Arg474X in exon 13 of the MTM1 gene, whereas the third patients with less pronounced manifestation, had a skewed pattern of X chromosome inactivation. DISCUSSION: Symptomatic female carriers of XLMTM can present with asymmetric malformations, which must be distinguished from an autosomal-dominant centronuclear myopathy. CONCLUSION: Unilateral presentation of weakness cannot rule out a diagnosis of myopathy. Detection of symptomatic female carriers of an X linked recessive disease, with a severe presentation in males, is important for genetic counselling.

Clinical, molecular, and genotype-phenotype correlation studies from 25 cases of oral-facial-digital syndrome type 1: a French and Belgian collaborative study.

Oral-facial-digital syndrome type 1 (OFD1) is characterised by an X linked dominant mode of inheritance with lethality in males. Clinical features include facial dysmorphism with oral, tooth, and distal abnormalities, polycystic kidney disease, and central nervous system malformations. Large interfamilial and intrafamilial clinical variability has been widely reported, and 18 distinct mutations have been previously reported within OFD1. A French and Belgian collaborative study collected 25 cases from 16 families. OFD1 was analysed using direct sequencing and phenotype-genotype correlation was performed using chi2 test. X inactivation studies were performed on blood lymphocytes. In 11 families, 11 novel mutations, including nine frameshift, one nonsense, and one missense mutation were identified, which spanned nine different exons. A combination of our results with previously reported cases showed that the majority of mutations (65.5%) was located in exons 3, 8, 9, 13, and 16. There was phenotype-genotype correlation between (a) polycystic kidney disease and splice mutations; (b) mental retardation and mutations located in exons 3, 8, 9, 13, and 16; and (c) tooth abnormalities and mutations located in coiled coil domains. Comparing the phenotype of the families with a pathogenic mutation to families with absence of OFD1 mutation, polycystic kidneys and short stature were significantly more frequent in the group with no OFD1 mutation, whereas lingual hamartomas were significantly more frequent in the group with OFD1 mutation. Finally, an X inactivation study showed non-random X inactivation in a third of the samples. Differential X inactivation between mothers and daughters in two families with high intrafamilial variability was of particular interest. Slight phenotype-genotype correlations were established, and X inactivation study showed that skewed X inactivation could be partially involved in the pathogenesis of intrafamilial clinical variability.

Mutations in EDAR account for one-quarter of non-ED1-related hypohidrotic ectodermal dysplasia.

Hypohidrotic ectodermal dysplasia (HED) is characterized by abnormal development of the eccrine sweat glands, hair, and teeth. The X-linked form of the disease, caused by mutations in the ED1 gene, represents the majority of HED cases. Autosomal-dominant and -recessive forms occur occasionally and result from mutations in at least two genes: EDAR and EDARADD. These different forms are phenotypically indistinguishable. To better assess the implication of the EDAR gene in HED, we screened for mutations in 37 unrelated HED families or sporadic cases with no detected mutations in the ED1 gene. We identified 11 different mutations, nine of which are novel variants, in two familial and seven sporadic cases. Seven of the 11 are recessive mutations (c.140G>A (p.Cys47Tyr), c.266G>A (p.Arg89His), c.329A>C (p.Asp110Ala), c.442T>C (p.Cys148Arg), c.1208C>T (p.Thr403Met), c.1302G>T (p.Trp434Cys) and c.528+1G>A), and the other four are probably dominant (c.1129C>T (p.Leu377Phe), c.1237A>C (p.Thr413Pro), c.1253T>C (p.Ile418Thr), and c.1259G>A (p.Arg420Gln)). Our study demonstrates that EDAR is implicated in about 25% of non-ED1 HED, and may account for both autosomal-dominant and -recessive forms. The correlation between the nature and location of EDAR mutations and their mode of inheritance is discussed. A genotype-phenotype relationship was evaluated, since such data could be helpful for genetic counseling.

Muscle involvement in limb-girdle muscular dystrophy with GMPPB deficiency (LGMD2T).

OBJECTIVE: In this study, muscle involvement assessed by MRI and levels of GMPPB and glycosylation of α-dystroglycan expression in muscle were examined in patients with limb-girdle muscular dystrophy (LGMD) type 2T. METHODS: Six new patients with genetically verified mutations in GMPPB were studied. T1-weighted magnetic resonance images were obtained in 4 participants. Muscle strength and potential involvement of extramuscular organs were examined. Glycosylation of α-dystroglycan in muscle was studied, and GMPPB and α-dystroglycan expression was analyzed by Western blotting. Prevalence of LGMD2T was calculated from the total LGMD population in Denmark. GMPPB was sequenced in all unclassified cases. RESULTS: Two patients carried 3 new mutations in GMPPB. The other 4 patients carried previously described pathogenic mutations in GMPPB. MRI showed that the paraspinal muscles were the most affected, followed by involvement of hamstrings. Our results showed a loss of glycosylation of α-dystroglycan as well as secondary loss of merosin expression on Western blotting. The prevalence of LGMD2T in the Danish cohort of patients with LGMD is 1.5%. CONCLUSIONS: The new findings of this study are (1) the consistent finding of a preferential affection of paraspinal and hamstring muscles in LGMD2T, (2) 3 new mutations in GMPPB, (3) variable loss of glycosylation tested with IIH6 and VIA4 antibodies, and (4) a prevalence of LGMD2T of 1.5% in a well-characterized Danish LGMD cohort.

[A clinical, neurophysiological and molecular study of 12 patients from 4 families with spinal and bulbar muscular atrophy]. / L'amyotrophie bulbaire et spinale liée au chromosome X: une étude clinique, neurophysiologique et moléculaire de 12 patients issus de 4 familles.

INTRODUCTION: Spinal and bulbar muscular atrophy (SBMA) is an X-linked, late-onset neuro-endocrine disorder resulting from an expansion of a CAG repeat in the androgen receptor gene. Material and method. We report the detailed phenotypic study in a series of 12 SBMA patients evaluated in four kindreds. RESULTS: Clinical phenotypic spectrum varied considerably, ranging from childhood-onset weakness and atrophy mimicking limb-girdle myopathy in patients with 53 CAG repeats to isolated hyperCKemia in an adult with 42 CAG repeats. All male patients had gynecomastia. Two female carriers presented with paresthesias and hand action tremor. Homozygous deletions of SMN1 and SMN2 genes were not found in any patients. CONCLUSION: This report demonstrates that SBMA may present with a wider clinical spectrum than previously described and suggests that clinical phenotype severity in SBMA is partially linked to the number of CAG repeats. It also suggests that SMN1 and SMN2 genes do not act as modifying genes in SBMA.

[Update on Bardet-Biedl syndrome]. / Le point sur le syndrome de Bardet-Biedl.

Until recently, Bardet-Biedl syndrome was considered as a classic autosomal recessive condition. The disorder is defined by the association of the following clinical features: retinitis pigmentosa, polydactyly, obesity, hypogonadism, and possible mental retardation. This syndrome leads to multiple handicaps (visual impairment, complications of obesity, kidney failure, endocrine dysfunction). This condition, apparently clearly defined from a clinical point of view, appears to be genetically heterogenous. To date, six different genes have been identified: BBS1, BBS2, BBS4, BBS6, BBS7 and BBS8. Interestingly, this condition has recently been linked to a failure of cellular ciliogenesis. Moreover, this disorder is characterized by an additional degree of complexity, as it is the first example of triallelic inheritance described in human beings. However, this new finding appears to be less frequent than expected in this syndrome.

Evidence for a common origin of most Friedreich ataxia chromosomes in the Spanish population.

Haplotype analysis is a powerful approach to understand the spectrum of mutations accounting for a disease in a homogeneous population. We show that haplotype variation for 10 markers linked to the Friedreich ataxia locus (FRDA) argues in favor of an important mutation homogeneity in the Spanish population, and positions the FRDA locus in the region where it has been recently isolated. We also report the finding of a new single nucleotide polymorphism called FAD1. The new marker shows a very strong linkage disequilibrium with Friedreich ataxia (FA) in both the Spanish and French populations. suggesting the existence of an ancient and widespread FRDA mutations. Inclusion of FAD1 in the extended haplotype analysis has allowed to postulate that this main FRDA mutation could account for 50-90% of the disease chromosomes. The results indicate that FA, despite clinical heterogeneity, could have originated from a few initial mutations.

[Paraneoplastic syndromes (1)]. / Les syndromes paranéoplasiques (1re partie).

Although not rare, paraneoplastic syndromes develop in a minority of cancer patients. Their exact frequency seems to be underestimated. These syndromes are hereby described. Their clinical usefulness is of great importance: they can be the first sign of a malignancy and can be used as tumor markers. The diagnosis of paraneoplastic syndromes is often difficult. A "diagnosis score" is proposed in order to make the diagnosis easier.

[Paraneoplastic syndromes (2)]. / Les syndromes paranéoplasiques (2e partie).

Although not rare, paraneoplastic syndromes develop in a minority of cancer patients. Their exact frequency seems to be under estimated. These syndromes are hereby described. Their clinical usefulness is of great importance: they can be the first sign of a malignancy and can be used as tumor markers. The diagnosis of paraneoplastic syndromes is often difficult. A "diagnosis score" is proposed to establish this diagnosis easier.

[Fragile X syndrome is still unrecognized: efficacy of molecular diagnosis in mentally retarded probands]. / Le syndrome X fragile est encore méconnu: efficacité du diagnostic moléculaire chez les proposants avec retard mental.

BACKGROUND: The fragile X mental retardation syndrome is the most common cause of inherited mental retardation. Identification of the unstable mutation responsible for the disease has allowed the design of a fully reliable molecular test for the diagnosis of the disease and for genetic counselling (identification of clinically normal carriers and prenatal diagnosis). We started in July 1991 to search for the mutation in mentally retarded probands, with no known cause for their phenotype. We present the results of a 42-month experience. POPULATION AND METHODS: One thousand and one hundred fourty-nine probands were analysed. In case of a positive diagnosis, an extension of the molecular study to relatives was proposed. DNA samples were studied by Southern blot following EcoRI or EcoRI + EagI digestion. Clinical data were collected from referring clinicians. RESULTS: Seventy-three carriers of a full mutation were identified, belonging to 52 families. The mean age of the fragile X probands was 16 +/- 14 years, which is very surprising for a disease that causes significant manifestations by the age of 2 to 3 years. This indicates an insufficient knowledge about this disease in France. Most of the demands for the test were from clinical geneticists. This diagnosis is of major importance for genetic counselling, as illustrated by the following study of 108 women at risk in these families. CONCLUSIONS: The importance of an early diagnosis followed by an extended family study, for carrier screening and prevention of this severe disease, justifies molecular testing on any child with mental retardation or significant language delay of unknown cause, in the absence of clinical signs formally excluding a fragile X diagnosis.

Dilated cardiomyopathy in patients with mutations in anoctamin 5.

BACKGROUND: Homozygous mutations in ANO5, a gene encoding anoctamin 5, a putative calcium-activated chloride channel, have recently been reported in patients with adult-onset myopathies or isolated high-CK levels. Cardiomyopathy has not previously been reported in these populations despite a proven expression of anoctamin 5 in the cardiac muscle. METHODS: Patients presenting for the management of high-CK levels or overt myopathy with proven ANO5 mutations were prospectively investigated between June 2010 and March 2012 in Pitié Salpêtrière Hospital, according to a standardised protocol. Neurological and cardiological clinical examinations, CK assessment, electrocardiogram (ECG), and echocardiography were performed, as well as cardiac MRI and coronary CT angiography in patients with left ventricular (LV) dysfunction. RESULTS: Our study included 19 consecutive patients (male=15, age=46.2 ± 12.7 years) from 16 families. Five had asymptomatic high-CK levels and 14 had overt myopathy. One patient had a personal history of stable coronary artery disease with normal ventricular function. ECG showed ventricular premature beats in one patient. Echocardiography displayed LV dilatation in two patients, LV dysfunction in one, and both abnormalities in two who fulfilled criteria for dilated cardiomyopathy which was confirmed by cardiac MRI and normal CT angiography. CONCLUSIONS: Dilated cardiomyopathy is a potential complication in patients with myopathies due to mutations in the ANO5 gene whose screening requires specific procedures.