Novel mutations in DNAJB6 cause LGMD1D and distal myopathy in French families.

BACKGROUND AND PURPOSE: The aim was to determine the genetic background of unknown muscular dystrophy in five French families. METHODS: Twelve patients with limb girdle muscular dystrophy or distal myopathy were clinically evaluated. Gene mutations were identified using targeted exon sequencing and mutated DNAJB6 was tested in vitro. RESULTS: Five patients presented with distal lower limb weakness whilst others had proximal presentation with a variable rate of progression starting at the mean age of 38.5 years. Two novel mutations (c.284A>T, p.Asn95Ile, two families; and c.293_295delATG, p.Asp98del, one family) as well as the previously reported c.279C>G (p.Phe93Leu, two families) mutation in DNAJB6 were identified. All showed a reduced capacity to prevent protein aggregation. CONCLUSIONS: The mutational and phenotypical spectrum of DNAJB6-caused muscle disease is larger than previously reported, including also dysphagia. The originally reported c.279C>G (p.Phe93Leu) mutation is now identified in four different populations and appears to be a mutational hotspot. Our report confirms that some DNAJB6 mutations cause distal-onset myopathy and hence DNAJB6 defects should be considered broadly in dominant muscular dystrophy families.

Association study reveals novel risk loci for sporadic inclusion body myositis.

BACKGROUND AND PURPOSE: The aim was to identify potential genetic risk factors associated with sporadic inclusion body myositis (sIBM). METHODS: An association based case-control approach was utilized on whole exome sequencing data of 30 Finnish sIBM patients and a control cohort (n = 193). A separate Italian cohort of sIBM patients (n = 12) was used for evaluation of the results. RESULTS: Seven single nucleotide polymorphisms were identified in five genes that have a considerably higher observed frequency in Finnish sIBM patients compared to the control population, and the previous association of the genetic human leukocyte antigen region was confirmed. CONCLUSIONS: All seven identified variants could individually or in combination increase the susceptibility for sIBM.

Targeted array comparative genomic hybridization--a new diagnostic tool for the detection of large copy number variations in nemaline myopathy-causing genes.

Nemaline myopathy (NM) constitutes a heterogeneous group of congenital myopathies. Mutations in the nebulin gene (NEB) are the main cause of recessively inherited NM. NEB is one of the most largest genes in human. To date, 68 NEB mutations, mainly small deletions or point mutations have been published. The only large mutation characterized is the 2.5 kb deletion of exon 55 in the Ashkenazi Jewish population. To investigate any copy number variations in this enormous gene, we designed a novel custom comparative genomic hybridization microarray, NM-CGH, targeted towards the seven known genes causative for NM. During the validation of the NM-CGH array we identified two novel deletions in two different families. The first is the largest deletion characterized in NEB to date, (∼53 kb) encompassing 24 exons. The second deletion (1 kb) covers two exons. In both families, the copy number change was the second mutation to be characterized and shown to have been inherited from one of the healthy carrier parents. In addition to these novel mutations, copy number variation was identified in four samples in three families in the triplicate region of NEB. We conclude that this method appears promising for the detection of copy number variations in NEB.

Eight new mutations and the expanding phenotype variability in muscular dystrophy caused by ANO5.

OBJECTIVE: Description of 8 new ANO5 mutations and significant expansion of the clinical phenotype spectrum associated with previously known and unknown mutations to improve diagnostic accuracy. METHODS: DNA samples of 101 patients in 95 kindreds at our quaternary referral center in Finland, who had undetermined limb-girdle muscular dystrophy (LGMD), calf distal myopathy, or creatine kinase (CK) elevations of more than 2,000 IU/L, were selected for ANO5 genetic evaluation, and the clinical findings of patients with mutations were retrospectively analyzed. RESULTS: A total of 25 patients with muscular dystrophy caused by 11 different recessive mutations in the ANO5 gene were identified. The vast majority of mutations, 8 of 11, proved to be previously unknown new mutations. The most frequent mutation, c.2272C>T (p.R758C), was present in 20 patients. The phenotypes associated with this and the common European mutation, c.191dupA, varied from nearly asymptomatic high hyperCKemia to severe LGMD with consistently milder phenotypes in female patients. CONCLUSIONS: Mutations in ANO5 are a frequent cause of undetermined muscular dystrophy, with both distal and proximal presentation. Other types include high hyperCKemia, myalgia, or calf hypertrophy over decades without significant weakness, especially in female patients. Mutations are distributed all over the gene, indicating that muscular dystrophy caused by ANO5 can be expected to occur in all populations.

Spanish MYH7 founder mutation of Italian ancestry causing a large cluster of Laing myopathy patients.

Laing myopathy is a distal myopathy caused by mutations in the tail of the slow beta-myosin heavy chain gene MYH7. A large cluster of patients belonging to different families, with Laing myopathy due to p.K1729del mutation, was found in the Safor region, Spain. The same mutation was previously reported in an American family with Italian ancestry. The possibility that p.K1729del in MYH7 might be a founder mutation in the Safor patients and the chance of a common origin with the Italian-American family mutation was investigated by haplotype analyses, mutation data origin estimation and historical inquiry. Our results show that the p.K1729del in MYH7 harboured by patients from the Safor indeed is a founder mutation. A common ancestral origin of this mutation in the Spanish and Italian families is also suggested because they all share a core SNP haplotype at locus MYH7. Data estimation yields the origin of the mutation in the Safor at the beginning of the XVII century, when the Moorish were spelt and the region was resettled with Italian families.

Late-onset lower motor neuronopathy: a new autosomal dominant disorder.

OBJECTIVE: Characterization of a new type of late-onset autosomal dominant lower motor neuron disease. METHODS: Patients from 2 families underwent detailed neurologic, electrophysiologic, muscle biopsy, and laboratory investigations. MRI of lower limbs was performed in selected patients. DNA samples from leukocytes were used for molecular genetic linkage studies. RESULTS: First symptoms were muscle cramps and fasciculations after age 25-30, followed by a slowly progressive proximal and distal weakness without overt atrophy during the first decades of symptoms. Nerve conduction velocities were within normal range and EMG showed widespread neurogenic alterations. Muscle biopsy revealed characteristic neurogenic findings: fiber type grouping and group atrophy. MRI showed diffuse fatty-degenerative changes, marked in medial gastrocnemius. CONCLUSION: Exactly the same clinical phenotype has not previously been described, and linkage studies showed exclusion of known chromosomal loci for hereditary motor neuropathies, suggesting the disease we report may represent a new disorder.

MYH7 gene tail mutation causing myopathic profiles beyond Laing distal myopathy.

OBJECTIVE: To describe a wide range of clinical and pathologic myopathic profiles associated with the p.K1729del mutation in the MYH7 gene, known to cause Laing distal myopathy. METHODS: A study conducted in the Safor region (Spain), setting of a large cluster of patients. Clinical, neurophysiologic, muscle imaging, and muscle biopsy studies and MYH7 gene sequencing were investigated in 32 patients from 4 kindreds. Data from 36 deceased or nonexamined patients were collected from hospital records or relatives. RESULTS: Onset ranged from congenital to the 6th decade. All patients presented weakness of great toe/ankle dorsiflexors and many had associated neck flexor, finger extensor, and mild facial weakness. In most cases, involvement of proximal and axial muscles was observed either clinically or by muscle imaging, sometimes giving rise to scapuloperoneal and limb-girdle syndromes. Disabling myalgias, skeletal deformities, and dilated cardiomyopathy in one patient were associated features. Life expectancy was not reduced but the spectrum of disability ranged from asymptomatic to wheelchair confined. Electromyographic neurogenic features were frequently recorded. Muscle fiber type disproportion, core/minicore lesions, and mitochondrial abnormalities were the most relevant pathologic alterations. All patients carried the p.K1729del mutation in MYH7. CONCLUSIONS: The p.K1729del mutation in the MYH7 gene expresses notable clinical variability and electromyographic and pathologic features that can lead to the misdiagnosis of neurogenic atrophies, congenital myopathies, or mitochondrial myopathies. Mutations in genes encoding other sarcomeric and reticulo-sarcoplasmic proteins involved in calcium regulation share pathologic characteristics with our patients, suggesting a possible pathogenetic connection.

Myopathies caused by homozygous titin mutations: limb-girdle muscular dystrophy 2J and variations of phenotype.

Limb-girdle muscular dystrophy 2J caused by mutations in C-terminal titin has so far been identified in Finnish patients only. This may in part be due to limited availability of diagnostic tests for titin defects. In this report, a French family with an autosomal-dominant late-onset distal myopathy of the tibial muscular dystrophy phenotype segregating in several members of the family was described. One deceased patient in the family proved to be homozygous for the C-terminal truncating titin mutation because of consanguinity. According to available medical records, the patient had a clearly more severe generalised muscle weakness and atrophy phenotype not recognised as a distal myopathy at the time. Autopsy findings in one of the original Finnish limb-girdle muscular dystrophy 2J patients were reported and the early phenotype in a newly identified young patient with homozygous Finnish C-terminal titin mutation (FINmaj) was detailed.

Zaspopathy in a large classic late-onset distal myopathy family.

Distal myopathies have been associated with mutations in titin, dysferlin, GNE, desmin and myosin. Of these, only titin mutations were previously known to cause dominant late-onset distal myopathy. Recent findings, however, have indicated that patients affected with myofibrillar myopathy have a more distal than proximal muscle phenotype and a proportion of these may have mutations in myotilin, ZASP or filamin C, besides previously known desmin and alphaB-crystallin. Here we report that the disorder in one of the well-characterized autosomal dominant distal myopathy families, the Markesbery et al. family, first reported in 1974, is caused by ZASP mutation A165V. Previous linkage to the titin locus 2q31 proved incorrect. ZASP expression by immunoblotting shows normal presence of the main 32 and 78 kDa bands and immunohistochemistry in patients reveals normal Z-disc localization except for moderate accumulations together with myotilin, desmin alphaB-crystallin and alpha-actinin. Muscle imaging reveals involvement in both the posterior and anterior compartments of the lower leg and considerable affection of proximal leg muscles at later stages. Haplotype studies in this family and in five other unrelated families with European ancestry carrying the identical A165V mutation share common markers at the locus suggesting the existence of a founder mutation.

Enrichment of the R77C alpha-sarcoglycan gene mutation in Finnish LGMD2D patients.

Limb-girdle muscular dystrophy 2D (LGMD2D) is caused by mutations in the alpha-sarcoglycan gene (SGCA). The most frequently reported mutation, 229CGC>TGC (R77C) in exon 3 of SGCA, results in the substitution of arginine by cysteine. We present here the clinical, immunohistochemical, and genetic data of 11 Finnish patients with LGMD2D caused by mutations in SGCA. Mutational analysis showed 10 patients homozygous and 1 compound heterozygous for R77C. A wide spectrum of SGCA mutations has been reported previously. Our results show an enrichment of R77C in Finland, further underlined by the observed carrier frequency of 1 per 150. According to the annual birth rate of approximately 60,000 in Finland, one LGMD2D patient with a homozygous mutation is expected to be born every 1 or 2 years on average. The presence of an ancient founder mutation is indicated by the fact that all patients shared a short common haplotype extending > or = 790 kilobases. Our results emphasize the need to include the SGCA gene R77C mutation test in routine DNA analyses of severe dystrophinopathy-like muscular dystrophies in Finland, and suggest that the applicability of this test in other populations should be studied as well.

Titinopathies and extension of the M-line mutation phenotype beyond distal myopathy and LGMD2J.

OBJECTIVE: To determine the phenotype variability associated with the specific C-terminal M-line titin mutation known to cause autosomal dominant distal myopathy, tibial muscular dystrophy (TMD; MIM 600334), and limb girdle muscular dystrophy 2J (LGMD2J). METHODS: Three hundred eighty-six individuals were genotyped for the Finnish founder mutation in titin (FINmaj) causing TMD/LGMD2J. RESULTS: Two hundred seven patients were heterozygous for the mutation. Among these patients, 189 (91%) had a more common phenotype compatible with the classic description of TMD. However, 18 (9%) had unusual phenotypes such as proximal leg or posterior lower leg muscle weakness and atrophy even at onset. Four patients were confirmed homozygotes representing the LGMD2J phenotype. These homozygotes were half of the eight LGMD patients previously described in the original large consanguineous kindred. CONCLUSIONS: Large variability of phenotypic expression caused by just one mutation, the Finnish FINmaj, suggests that no certain phenotype of myopathy/dystrophy can be excluded from being caused by mutated titin. Yet unknown homozygous or compound heterozygous titin mutations without phenotype in the heterozygote carriers may be responsible for undetermined recessive MD and LGMD.

POLG mutations in neurodegenerative disorders with ataxia but no muscle involvement.

OBJECTIVE: To identify POLG mutations in patients with sensory ataxia and CNS features. METHODS: The authors characterized clinical, laboratory, and molecular genetic features in eight patients from five European families. The authors conducted sequencing of coding exons of POLG, C10orf2 (Twinkle), and ANT1 and analyzed muscle mitochondrial DNA (mtDNA), including Southern blot analysis and long-range PCR. RESULTS: Ataxia occurred in combination with various CNS features, including myoclonus, epilepsy, cognitive decline, nystagmus, dysarthria, thalamic and cerebellar white matter lesions on MRI, and neuronal loss in discrete gray nuclei on autopsy. Gastrointestinal dysmotility, weight loss, cardiomyopathy, and valproate-induced hepatotoxicity occurred less frequently. Two patients died without preceding signs of progressive external ophthalmoplegia. In muscle, typical findings of mitochondrial disease, such as ragged red fibers and Southern blot mtDNA abnormalities, were absent. POLG mutations were present in eight patients, including two isolated cases, and one Finnish and two unrelated Belgian families contained in total six patients. All POLG mutations were recessive, occurring in a homozygous state in seven patients and in a compound heterozygous state in one patient. The novel W748S mutation was identified in five patients from three unrelated families. CONCLUSIONS: The clinical spectrum of recessive POLG mutations is expanded by sensory ataxic neuropathy, combined with variable features of involvement of CNS and other organs. Progressive external ophthalmoplegia, myopathy, ragged red fibers, and Southern blot abnormalities of muscle mitochondrial DNA also are not mandatory features associated with POLG mutations.

Muscle magnetic resonance imaging shows distinct diagnostic patterns in Welander and tibial muscular dystrophy.

OBJECTIVES: This is a report on a retrospective muscle magnetic resonance imaging (MRI) study on 11 patients affected by Welander distal myopathy (WDM) and 22 patients with tibial muscular dystrophy (TMD) carried out in order to define the pattern and characteristics of muscle involvement. RESULTS: WDM patients showed involvement of gastrocnemius, soleus, tibial anterior (TA) and extensor digitorum longus (EDL), as well as hamstrings and hip adductor muscles. TMD patients showed involvement of the TA and EDL muscles, and in some patients also hamstring and posterior compartment muscles of the legs. Some patients showed asymmetry of muscle involvement. CONCLUSION: We conclude that muscle MRI examination proved to be very useful in the determination of the exact pattern of muscle involvement in WDM and TMD. Clinical testing using the Medical Research Council scale is not sensitive enough to establish the pattern of muscle involvement in focal muscle diseases.

New methods for molecular diagnosis and demonstration of the (CCTG)n mutation in myotonic dystrophy type 2 (DM2).

Myotonic dystrophy types 1 and 2 are autosomal dominant, multisystemic disorders with many similarities in their clinical manifestations. Myotonic dystrophy type 1 is caused by a (CTG)n expansion in the 3' untranslated region of the DMPK gene in 19q13.3 and myotonic dystrophy type 2 by a (CCTG)n expansion in intron 1 of ZNF9 in 3q21.3. However, the clinical diagnosis of myotonic dystrophy type 2 is more complex than that of myotonic dystrophy type 1, and conventional molecular genetic methods used for diagnosing myotonic dystrophy type 1 are insufficient for myotonic dystrophy type 2. Herein we describe two in situ hybridization protocols for the myotonic dystrophy type 2 mutation detection. Chromogenic in situ hybridization was used to detect both the genomic expansion and the mutant transcripts in muscle biopsy sections. Chromogenic in situ hybridization can be used in routine myotonic dystrophy type 2 diagnostics. Fluorescence in situ hybridization on extended DNA fibers was used to directly visualize the myotonic dystrophy type 2 mutation and to estimate the repeat expansion sizes.

Tibial muscular dystrophy in a Belgian family.

We report a Belgian family with autosomal dominant, late-onset, distal myopathy with selective foot extensor muscle involvement of the lower legs. Linkage to the tibial muscular dystrophy (TMD) locus 2q31 was not evident at first because of incomplete disease penetrance in a 50-year-old asymptomatic family member. An abnormal tibialis anterior muscle biopsy established her subclinical status and linkage of the family to the TMD locus. Mutation analysis showed a disease-specific, heterozygous point mutation in the last exon, Mex6, of the titin gene. This is the third mutation found in TMD and the second European family with TMD outside the Finnish population, suggesting that titinopathies may occur in various populations.

Congenital myasthenic syndrome associated with episodic apnea and sudden infant death.

The sudden infant death syndrome has multiple etiologies. Some congenital myasthenic syndromes can cause sudden infant death syndrome by apnea, but the frequency of this etiology is unknown. We report here a young patient with sudden respiratory crises culminating in apnea followed by recovery, against a background of no or variable myasthenic symptoms without dyspnea. One sib without myasthenic symptoms and one sib who only had mild ptosis died previously during febrile episodes. Studies reported by us elsewhere traced the proband's illness to mutations in choline acetyltransferase. Here, we describe in detail the morphologic investigations and electrophysiologic findings, which point to a presynaptic defect in acetylcholine resynthesis or vesicular filling, in the proband. Analysis of DNA from a sib who previously died of sudden infant death syndrome revealed the same choline acetyltransferase mutation. Thus, mutations in choline acetyltransferase may be a cause of sudden infant death syndrome as, theoretically, could other presynaptic myasthenic disorders.

Recognizing and understanding the cold-stressed term infant.

Hypothermia is an important cause of morbidity--and occasionally mortality--in the newborn. This article explains the physiology of thermoregulation and thermogenesis in term infants. Subjects include the role of the hypothalamus in heat production, causes of and ways to prevent heat loss, and establishment of a neutral thermal environment. Complications associated with cold stress and thcir etiologies, as well as methods for rewarming an infant, are also addresscd.

Mutational spectra at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in T-lymphocytes of nonsmoking and smoking lung cancer patients.

Molecular analysis of mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in peripheral blood T-lymphocytes can provide information on mechanisms of somatic in vivo mutation in populations exposed to exogenous carcinogens and in individuals with inherent susceptibility to cancer and other diseases. To study possible mutational changes associated with smoking as a risk factor for lung cancer, we analyzed HPRT mutations in T-cells of newly diagnosed, nonsmoking and smoking lung cancer patients before treatment. Reverse transcriptase polymerase chain reaction (RT-PCR) and DNA sequencing methods were used to identify 146 independent mutations, 73 each from 32 nonsmoking and 31 smoking cases. In 35 T-cell mutants, the HPRT cDNA showed loss of an entire exon, indicating a splicing mutation. Among the remaining 111 fully characterized mutations in the coding region, single base pair (bp) substitutions predominated with 79% (48/61) in nonsmokers and 90% (45/50) in smokers. Frameshift and small deletion (1-24 bp) mutations were found in 18 mutants. The distribution of base pair substitutions was nonrandom, with significant clustering at previously identified hotspot positions 143, 197 and 617 in the HPRT coding sequence (P< or =0.008). One additional hotspot, GC-->TA at position 606, was observed only in smokers (P=0.006). The frequency of GC>TA transversions was higher in smokers (13%) than in nonsmokers (6%). Conversely, smokers had a lower frequency of GC>AT transitions (24%) than nonsmokers (35%). This smoking-associated shift of the HPRT mutational spectrum, although not statistically significant, is consistent with the in vitro mutagenicity of benzo(a)pyrene (BaP), a prominent carcinogen of tobacco smoke, and with known differences in the TP53 mutational spectrum in lung tumors of smokers and nonsmokers. Among nonsmokers, the HPRT mutational spectra in healthy population controls and lung cancer patients were similar, but there was a marginally significant difference (P=0.07) in the distribution of base pair substitutions between smoking controls and patients. These results suggest that (i) general mechanisms of somatic mutagenesis in individuals with possible predisposition to cancer (e.g. nonsmoking lung cancer patients) are not different from those in normal healthy individuals, and (ii) the HPRT gene in T-cells is a useful reporter locus for smoking-associated somatic in vivo mutations occurring early in lung cancer development.

Missense mutations in hMLH1 associated with colorectal cancer.

One of the most prevalent hereditary syndromes associated with colorectal cancer is hereditary nonpolyposis colorectal cancer (HNPCC). The inherited gene defects in HNPCC have been shown to reside in DNA mismatch repair genes, mostly hMSH2 or hMLH1. Most HNPCC patients are heterozygous with regard to the relevant mismatch repair gene; they have one normal and one mutated allele, and mismatch repair in normal somatic cells is functional. Cancer predisposition in HNPCC is believed to be associated with the loss of the wild-type allele in somatic cells, resulting in defective DNA mismatch repair. This gives rise to DNA microsatellite instability (MSI), an increased somatic mutation rate, and eventually, to the accumulation of mutations in genes involved in colorectal carcinogenesis. In support of this theory, colorectal tumors in HNPCC patients and in mice deficient for hMSH2 or hMLH1 show MSI. Here, we describe two missense mutations in hMLH1 exon 16 associated with colorectal cancer. Interestingly, the tumors do not show MSI. This raises some potentially important issues. First, even microsatellite-negative colorectal tumors can be associated with germline mutations and these will be missed if an MSI test is used to select patients for mutation screening. Second, the lack of MSI in these cases suggests that the mechanism involved in carcinogenesis could be different from that generally hypothesized.

Radiation induced chromosomal instability in human T-lymphocytes.

Chromosomal instability in proliferating mammalian cells is characterized by a persistent increase of chromosomal aberrations and rearrangements occurring de novo during successive cell generations. Recent results from many laboratories using a variety of cells and cytogenetic end points show that this phenotype can be induced by low as well as high LET irradiation. A typical feature of chromosomal instability in primary human G0-lymphocytes exposed to gamma-irradiation at both high dose rate (45 Gy h-1) and low dose rate (0.024 Gy h-1) is the appearance of novel aberrations in the clonal progeny of the irradiated cell, many generations after the exposure. The same phenotype was observed in lymphocytes that were allowed to recover for 5 days in G0 after the radiation exposure, as well as in hprt-mutant T cell clones. These results demonstrate that neither the acute genotoxic stress caused by high dose rate as compared to low dose rate irradiation, nor a hypothesized conflict between mitogen induced growth stimulation and growth arrest due to radiation damage, seem to be critical conditions for the development chromosomal instability in these cells. In contrast to observations in other cells, no evidence of a persistent decrease of cloning ability was observed in the progeny of radiation-exposed human lymphocytes, and no alteration was observed in their sensitivity to a second radiation exposure. Furthermore, the frequency of CA-repeat length variation at three loci was not increased in the progeny of X-irradiated T cells as compared to non-irradiated cells, which indicates that microsatellite instability is not part of the chromosomal instability phenotype in human T-lymphocytes.