Scapular dyskinesis in myotonic dystrophy type 1: clinical characteristics and genetic investigations.

OBJECTIVE: To study scapular winging or other forms of scapular dyskinesis (condition of alteration of the normal position and motion of the scapula) in myotonic dystrophy type 1 (DM1), which is generally considered to be a distal myopathy, we performed an observational cohort study. METHODS: We performed a prospective cohort study on the clinical features and progression over time of 33 patients with DM1 and pronounced, mostly asymmetric scapular winging or other forms of scapular dyskinesis. We also explored if scapular dyskinesis in DM1 has the same genetic background as in facioscapulohumeral muscular dystrophy type 1 (FSHD1). RESULTS: The cohort included patients with congenital (n = 3), infantile (n = 6) and adult-onset DM1 (n = 24). Scapular girdle examination showed moderate shoulder girdle weakness (mean MRC 3) and atrophy of trapezius, infraspinatus, and rhomboid major, seemingly similar as in FSHD1. Shoulder abduction and forward flexion were limited (50-70°). In five patients, scapular dyskinesis was the initial disease symptom; in the others it appeared 1-24 years after disease onset. Follow-up data were available in 29 patients (mean 8 years) and showed mild to severe increase of scapular dyskinesis over time. In only three patients, DM1 coexisted with a FSHD mutation. In all other patients, FSHD was genetically excluded. DM2 was genetically excluded in nine patients. The clinical features of the patients with both DM1 and FSHD1 mutations were similar to those with DM1 only. CONCLUSION: Scapular dyskinesis can be considered to be part of DM1 in a small proportion of patients. In spite of the clinical overlap, FSHD can explain scapular dyskinesis only in a small minority. This study is expected to improve the recognition of shoulder girdle involvement in DM1, which will contribute to the management of these patients.

Common SNPs of AmelogeninX (AMELX) and dental caries susceptibility.

Genetic approaches have shown that several genes could modify caries susceptibility; AmelogeninX (AMELX) has been repeatedly designated. Here, we hypothesized that AMELX mutations resulting in discrete changes of enamel microstructure may be found in children with a severe caries phenotype. In parallel, possible AMELX mutations that could explain resistance to caries may be found in caries-free patients. In this study, coding exons of AMELX and exon-intron boundaries were sequenced in 399 individuals with extensive caries (250) or caries-free (149) individuals from nine French hospital groups. No mutation responsible for a direct change of amelogenin function was identified. Seven single-nucleotide polymorphisms (SNPs) were found, 3 presenting a high allele frequency, and 1 being detected for the first time. Three SNPs were located in coding regions, 2 of them being non-synonymous. Both evolutionary and statistical analyses showed that none of these SNPs was associated with caries susceptibility, suggesting that AMELX is not a gene candidate in our studied population.

Bisulfite genomic sequencing of microdissected cells.

Mapping of methylation patterns in CpG islands has become an important tool for understanding tissue-specific gene expression in both normal and pathological situations. However, the inherent cellular heterogeneity of any given tissues can affect the outcome and interpretation of molecular studies. In order to analyse genomic DNA methylation on a pure cell population from tissue sample, we have developed a simple technique of single-cell microdissection from cryostat sections which can be combined with bisulfite-mediated sequencing of 5-methylcytosine. We report here our results on the methylation status of the androgen receptor gene studied by bisulfite genomic sequencing on purified cells isolated from human testis.

[Diagnostic trap and difficulties of genetic counseling in a family with neuromuscular disease carriers]. / Piège diagnostique et difficultés du conseil génétique dans une famille de patients porteurs de maladies neuromusculaires.

BACKGROUND: Recent advances in the field of molecular genetics have provided useful tools for the diagnosis of neuromuscular disorders. Genetic counselling for many of these conditions may, however, be fraught with difficulties. CASE REPORT: The patient, two paternal uncles and a paternal aunt presented with clinical and electromyographic evidence of type III spinal muscular atrophy despite an autosomal dominant-like pedigree. The diagnosis was confirmed by genetic testing for the SMN deletion. As the proband's mother was pregnant at the time of presentation of the affected child, a prenatal diagnostic test was performed. The deletion was not found in the DNA extracted from the trophoblast and the pregnancy proceeded to full term, and a normal child. At the same time, a first cousin of the proband was found to have a clinically similar condition. He had not the SMN deletion. He presented with electrophysiological and pathological features of limb-girdle muscular dystrophy. Genetic testing revealed a homozygote del T521 mutation of the gama-sarcoglycan gene. CONCLUSION: To provide accurate genetic counselling, it is essential to get precise data on family background and diagnostic confirmation for each affected relative to avoid missing the possibility, albeit rare, of several neuromuscular disorders within a family.

Improved characterization of FSHD mutations.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the shortest alleles of the 3.3kb-tandem repeat array D4Z4 at 4q35. Molecular diagnosis of FSHD depends upon the separation of unusually large alleles by pulse-field electrophoresis after EcoRI and EcoRI/BlnI digestion. The exact number of alleles could not however be directly inferred from the size of DNA fragments owing to polymorphisms in the telomeric region of the locus. Knowing the exact repeat number of disease causing alleles may benefit genetic counselling, help to understand the mechanism of this singular disease and the population dynamics of subtelomeric sequences variations. We present here a partial digestion mapping method giving the exact number of repeats for disease causing alleles, and we suggest that most inaccuracies induced by common polymorphisms could be reduced by using EcoRV in place of EcoRI. After studying more than 300 DNA samples with both the standard method and this new method, we show that alleles size can be evaluated with a precision of less than one half repeat, and that the variations in length of the truncated repeat in the telomeric region of the D4Z4 locus can be evaluated. The results suggest that at least one intact chromosome 4 type repeat at 4q35 is needed to cause FSHD.

The photoreceptor cell-specific nuclear receptor gene (PNR) accounts for retinitis pigmentosa in the Crypto-Jews from Portugal (Marranos), survivors from the Spanish Inquisition.

The last Crypto-Jews (Marranos) are the survivors of Spanish Jews who were persecuted in the late fifteenth century, escaped to Portugal and were forced to convert to save their lives. Isolated groups still exist in mountainous areas such as Belmonte in the Beira-Baixa province of Portugal. We report here the genetic study of a highly consanguineous endogamic population of Crypto-Jews of Belmonte affected with autosomal recessive retinitis pigmentosa (RP). A genome-wide search for homozygosity allowed us to localize the disease gene to chromosome 15q22-q24 (Zmax=2.95 at theta=0 at the D15S131 locus). Interestingly, the photoreceptor cell-specific nuclear receptor (PNR) gene, the expression of which is restricted to the outer nuclear layer of retinal photoreceptor cells, was found to map to the YAC contig encompassing the disease locus. A search for mutations allowed us to ascribe the RP of Crypto-Jews of Belmonte to a homozygous missense mutation in the PNR gene. Preliminary haplotype studies support the view that this mutation is relatively ancient but probably occurred after the population settled in Belmonte.

Sarcoglycanopathies in Dutch patients with autosomal recessive limb girdle muscular dystrophy.

Within a group of 76 sporadic/autosomal recessive limb girdle muscular dystrophy (LGMD) patients we tried to identify those with LGMD type 2C-E. Muscle biopsy specimens of 40 index patients, who had 22 affected sibs, were analyzed immuno-histochemically for the presence of three subunits: alpha-, beta-, and gamma-sarcoglycans. Abnormal sarcoglycan expression was established in eight patients, with six affected sibs. In one patient gamma-sarcoglycan was absent, and both alpha- and beta-sarcoglycans were reduced. In the remaining seven patients gamma-sarcoglycan was (slightly) reduced, and alpha- and beta-sarcoglycans were absent or reduced. By DNA sequencing mutations were detected in one of the three sarcoglycan genes in all eight cases. Three patients had mutations in the alpha-, three in the beta-, and two in the gamma-sarcoglycan gene. The patients with sarcoglycanopathy comprised the more severely affected cases (P=0.04). In conclusion, sarcoglycanopathy was identified in 23 % (14/62) of the autosomal recessive LGMD patients.

Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis.

Parental-specific epigenetic modifications are imprinted on a subset of genes in the mammalian genome during germ cell maturation. However, the precise timing of their establishment remains to be determined. Methylation of CpG dinucleotides has been shown to be a part of the parental imprint. We have examined how the methylation pattern characteristic of the paternal allele in germ cells are established during human spermatogenesis. Two representative imprinted genes, H19 and MEST/PEG1, were studied. The experiments were performed using the bisulphite sequencing method on microdissected individual cells at different stages of male germ cell differentiation. We show that both genes are unmethylated in fetal spermatogonia, suggesting that all pre-existing methylation imprints are already erased by this stage. The MEST/PEG1 gene remains unmethylated at all subsequent post-pubertal stages of spermatogenesis, including mature spermatozoa. The methylation of H19 typical of the paternal allele first appears in a subset of adult spermatogonia and then is maintained in spermatocytes, spermatids and mature spermatozoa. Our results suggest that the methylation imprint inherited from the parents is first erased in the male germ line at an early fetal stage. The paternal-specific imprint is re-established only later, during spermatogonial differentiation in the adult testis.

Homogeneous phenotype of the gypsy limb-girdle MD with the gamma-sarcoglycan C283Y mutation.

OBJECTIVE: To characterize the clinical phenotype of LGMD2C in gypsies. BACKGROUND: Limb-girdle muscular dystrophy (LGMD) in gypsies of Western Europe is caused by a homozygous C283Y mutation on the same haplotype, suggesting a founder effect. METHODS: We performed clinical, laboratory, and muscle imaging studies of 40 patients. RESULTS: Mean age at onset was 5.3 years. One half of the patients had loss of ambulation by the age of 12; 13% still could walk after age 16. Calf hypertrophy, scapular winging, macroglossia, and lumbar hyperlordosis were common. Girdle, trunk, and proximal limb flexor muscles had earlier and more severe involvement. Cardiomyopathy was not observed. Five patients in the third decade of life required mechanical ventilation. Scoliosis was common in the nonambulatory stage. CONCLUSIONS: LGMD2C in gypsy patients with C283Y mutation presents a rather homogeneous phenotype, characterized by an initial Duchenne-like progressive course followed by a more prolonged survival rate possibly due to the absence of early respiratory impairment and cardiac failure.

Fluorescence in situ hybridization analysis of chromosome 1 abnormalities in hematopoietic disorders: rearrangements of DNA satellite II and new recurrent translocations.

Using fluorescence in situ hybridization analysis, breakpoints involving the long arm of chromosome 1 (1q) were localized in 36 patients with various hematopoietic disorders and rearrangements of the proximal part of 1q, as ascertained with banding techniques. The breakpoint was localized within the satellite II (sat II) domain in 14 patients with various abnormalities, between the sat II domain and the BCL9 locus in eight, between the BCL9 and ARNT loci in two, between sat II and ARNT in two others, and distal to ARNT in seven. A dicentric chromosome 1 was present in two patients. A high incidence of heterochromatin heteromorphism of chromosome 1 was present in this series. Two recurrent translocations were identified, t(1;2)(q12;q37) in three patients suffering from three different acute leukemia subtypes, and t(1;16)(q12;q24) in two patients with different diseases. Two patients had jumping translocations. Most of the rearrangements of 1q were secondary abnormalities, included in complex karyotypes. The roles of methylation, interactions with the proteins interfering with heterochromatin and possible gene silencing due to heterochromatin rearrangements are discussed.

Abnormal methylation does not prevent X inactivation in ICF patients.

DNA undermethylation is a characteristic feature of ICF syndrome and has been implicated in the formation of the juxtacentromeric chromosomal abnormalities of this rare syndrome. We have previously shown that in female ICF patients the inactive X chromosome (Xi) is also undermethylated. This result was unexpected since female ICF patients are not more severely affected than male patients. Here we show that CpG island methylation is abnormal in some ICF patients but in other ICF patients, the difference in methylation pattern between Xi and Xa (active X) is maintained. The consequences of Xi undermethylation on gene expression were investigated by enzyme assays. They showed that significant gene expression did not correlate with CpG island methylation status. The widespread Xi undermethylation does not affect overall Xi replication timing and does not prevent Barr body formation suggesting that a normal methylation pattern is not required for normal chromatin organization of Xi. Molecular investigation of some X-chromosome intron regions showed that the methylation changes in ICF female patients extend to non CpG islands sequences. Our results suggest that the genetic alteration of DNA methylation in ICF syndrome has little consequence on X chromosome gene expression and chromatin organization.

[Genes and intra-uterine growth retardation]. / Gènes et retards de croissance intra-utérins.

Genetic analysis of growth from birth to adulthood shows the existence of a strong genetic component in the variance of size at particular milestones in the growth process, such as height at take-off, at peak velocity and at adulthood. While it is well known that postnatal growth is strongly genetically determined, the importance of genes in normal variations of prenatal growth is less known. Genetic analysis of prenatal growth in human is not an easy problem and weighing the genetic and non-genetic component in intra-uterine growth retardation an almost impossible task. It is now well known that adults who had a low birthweight or who were thin at birth, with a low ponderal index, tend to be insulin resistant and have an increased risk of developing non-insulin-dependent diabetes mellitus later in life. According to the thrifty genotype hypothesis, genes predisposing to type 2 diabetes mellitus are very likely to have been survival genes for our ancestors, helping them to store energy during long periods of starvation. Both epidemiological surveys of adults born after prenatal exposure to exposure to famine and biochemical investigations of insulin resistance in low birthweight children show that the association between a low birthweight and an increased risk of developing type 2 diabetes mellitus later in life has a genetic basis. While low birthweight infants have a decreased survival probability in infancy, having a small baby may have been a selective advantage during long periods of starvation. This could explain why the same genetic variants cause low birthweight phenotype and insulin resistance predisposing to non-insulin-dependent diabetes.

Cardiac involvement in genetically confirmed facioscapulohumeral muscular dystrophy.

In a series of 100 patients exhibiting clinical and molecular features of facioscapulohumeral muscular dystrophy (FSHMD), five patients had conduction defects or arrhythmia in the absence of cardiovascular risk factors--namely, intraventricular conduction delay and supraventricular arrhythmia induced by electrophysiologic investigations (two patients), palpitations associated with supraventricular arrhythmia (one patient), severe atrioventricular block leading to pacemaker implantation (one patient), and ventricular tachycardia related to arrhythmogenic right ventricular cardiomyopathy (one patient). Patients with FSHMD may have cardiac involvement.

Severe limb girdle muscular dystrophy in Spanish gypsies: further evidence for a founder mutation in the gamma-sarcoglycan gene.

Limb-girdle muscular dystrophy type 2C (LGMD2C) is an autosomal recessive muscular dystrophy with primary gamma-sarcoglycan deficiency, generally associated with a severe clinical course. gamma-sarcoglycan, a 35kDa dystrophin-associated protein, is encoded by a single gene on chromosome 13q12. Six different mutations have been described in that gene, and it has been proved they are the origin of the disease. One of these mutations (C283Y), a G-->A transition in codon 283, was recently and exclusively identified in Gypsy patients from different European countries. We report the study of 11 LGMD2C unrelated Gypsy families (nine Spanish and two Portugese). The muscle biopsies of these patients showed a drastically decreased immunostaining with alpha and gamma-sarcoglycan antibodies. All the patients were homozygous for C283Y missense mutation, and all affected chromosomes (patients and heterozygous relatives) carried the allele 5 (112 bp) of the intragenic microsatellite D13S232. Unexpectedly, this allele is most frequent in the Caucasian population but not in the normal Gypsy population. The clinical severity of all patients demonstrates that the C283Y missense mutation in a homozygous state causes a severe LGMD2C (DMD-like). The elevated number of families ascertained let us assume that LGMD2C is prevalent in the Gypsy population, and that all the families have inherited a founding mutation.

Prenatal diagnosis of limb-girdle muscular dystrophy type 2C.

After studies which have mapped the gamma-sarcoglycan deficient limb-girdle muscular dystrophy (LGMD2C) to chromosome 13q12 and recent identification of mutations within this gene, prenatal diagnosis has become possible. The deletion of exon 5 in the gamma-sarcoglycan gene was found in a consanguineous family and prenatal diagnosis was successfully provided. This is the first prenatal diagnosis of LGMD2C.

A biochemical, genetic, and clinical survey of autosomal recessive limb girdle muscular dystrophies in Turkey.

Autosomal recessive limb girdle muscular dystrophy (LGMD2) is a clinically and genetically heterogenous group of diseases involving at least six different loci. Five genes have already been identified: calpain-3 at LGMD2A (15q15), and four members of the sarcoglycan (SG) complex, alpha-SG at LGMD2D (17q21), beta-SG at LGMD2E (4q12), gamma-SG at LGMD2C (13q12), and delta-SG at LGMD2F (5q33-q34). The gene product at LGMD2B (2p13-p16) is still unknown and at least one other gene is still unmapped. We investigated 20 Turkish families (18 consanguineous) diagnosed as having LGMD2. Most of our patients had onset of symptoms before age 10. The phenotypes varied from severe to benign. We analyzed the SG complex by immunofluorescence and/or western blot. Genotyping was performed using markers defining the six known loci and the suspected genes were screened for mutations. Six of 17 index cases showed deficiency of the SG complex, by immunofluorescence and/or western blot. Seven cases involved one of the known genes of the SG complex (alpha, 2; beta, 1; and gamma, 4 cases), and five mutations were documented in the alpha- and gamma-SG genes. After linkage analysis, 10 families were characterized as having LGMD2A (calpain-3 deficiency), and all mutations were eventually identified. One family was classified as having LGMD2B and 1 family that has normal SGs was linked to the chromosome 5q33-q34 locus (LGMD2F). In 1 family there was no linkage to any of the known LGMD2 loci. It appears that in Turkey, there is a broad spectrum of genes and defects involved in LGMD2. It may be possible to correlate genotype to phenotype in LGMD2. All severe cases belonged to the gamma-SG-deficiency group. Nine calpain-3-deficient cases had intermediate and 1 had moderate clinical courses. The LGMD2B patient had a moderate clinical expression, whereas the LGMD2F case was truly benign.

Mutational diversity and hot spots in the alpha-sarcoglycan gene in autosomal recessive muscular dystrophy (LGMD2D).

Sarcoglycanopathies are a genetically heterogeneous group of autosomal recessive muscular dystrophies in which the primary defect may reside in any of the genes coding for the different partners of the sarcolemmal sarcoglycan (SG) complex: the alpha-SG (LGMD2D at 17q21.2), the beta-SG (LGMD2E at 4q12), the gamma-SG (LGMD2C at 13q12), and the delta-SG (LGMD2F at 5q33). We report a series of 20 new unrelated families with 14 different mutations in the alpha-SG gene. Along with the mutations that we previously reported this brings our cohort of patients with alpha-sarcoglycanopathy to a total of 31 unrelated patients, carrying 25 different mutations. The missense mutations reside in the extracellular domain of the protein. Five of 15 missense mutations, carried by unrelated subjects on different haplotype backgrounds and of widespread geographical origins, account for 58% of the mutated chromosomes, with a striking prevalence of the R77C substitution (32%). The severity of the disease varies strikingly and correlates at least in part with the amount of residual protein and the type of mutation. The recurrent R284C substitution is associated with a benign disease course.

alpha-satellite DNA methylation in normal individuals and in ICF patients: heterogeneous methylation of constitutive heterochromatin in adult and fetal tissues.

The methylation profile of ten alpha-satellites was investigated in normal individuals and in ICF (Immunodeficiency, Centromeric instability, Facial abnormalities) patients. Two out of three ICF patients showed modified methylation of these sequences, reproducing a placental profile. CENP-B boxes, the binding sites of centromeric protein B, were always skewed toward nonmethylation. Unexpected results were observed in normal individuals: in somatic adult tissues the methylation pattern of alpha-satellite DNA varied between chromosomes, and in fetal tissues these satellites were homogeneously undermethylated. Detailed methylation analysis of CENP-B boxes revealed that unmethylated alpha-satellite units coexist with thoroughly methylated regions. These observations showed that the two major components of constitutive heterochromatin are differently methylated in normal somatic and fetal tissues, since classical satellites are consistently methylated. The definite changes in the methylation profile of heterochromatin in somatic chromosomes and the asynchronous timing of methylation of classical and alpha-satellites during development may reflect specific roles of highly repeated sequences in genomic organization.