Expansion of CTG repeat in myotonin protein kinase gene on Alu(ins)‐Hinf1‐1 background in a myotonic dystrophy patient from India

To determine the founder of Indian myotonic dystrophy mutation, we have studied the expansion of CTG repeats in myotonin protein kinase gene and two intragenic linked loci Alu(ins) / Alu(del) and G/T intron 9 Hinf1 polymorphism in ten unrelated DM patients from eastern India. Out of these ten patients, reconstruction of haplotype was possible for five patients unambiguously. In the other five cases, haplotype for the normal allele was assumed to be the most common haplotype found in normal individuals from Indian populations. Such analysis showed that in nine cases, the expansion of CTG repeats took place on Alu(ins)‐Hinf1‐2 background indicating common founder with other DM mutation published. However, in one case we observed a different haplotype [Alu(ins)‐Hinf1‐1] which could be a new mutation or due to admixture. © 1998 Wiley‐Liss, Inc.     

Molecular anatomy of CTG expansion in myotonin protein kinase gene among myotonic dystrophy patients from eastern India

We have studied the CTG repeat sizes in the DMPK gene and six biallelic markers which are in complete linkage disequlibrium with Caucasian DM patients, to identify any common founder haplotype in 30 clinically diagnosed unrelated DM patients from eastern India. Our results revealed that in 27 patients (90%), CTG expansion took place on a DraIII(-) - HhaI(-) - Alu(+) - HinfI(+) - Fnu4H I(-) - TaqI(+) haplotype (haplotype I), similar to what have been published for Caucasoid and other DM patients. However, in three patients (10%), the expansion of CTG repeat was on DraIII(+) - HhaI(+) - Alu(+) - HinfI(-) - Fnu4H I(+) - TaqI(-) background (haplotype II), indicating a new haplotype. The distribution of haplotypes in 52 normal individuals of eastern India revealed that percentage of haplotypes I and II were 23.1% and 7.7% respectively in normal chromosomes. Haplotype II is absent among Caucasian DM patients as well as normal individuals indicating that this particular haplotype may be characteristic of the Indian population. Hum Mutat 16:372, 2000.     

High CTG repeat number in nodular thyroid tissue from a myotonic dystrophy patient.

CTG triplet expansion was studied in lymphocytes and thyroid tissue in a patient with myotonic dystrophy (DM) and associated thyroid nodular disease. An approximately 7 fold larger amplification was found in abnormal thyroid tissue compared to lymphocytes, suggesting that anomalies in the putative DM kinase gene might contribute to thyroid dysfunction.     

Novel isoform of myotonin protein kinase: gene product of myotonic dystrophy is localized in the sarcoplasmic reticulum of skeletal muscle.

It is quite important to know the exact localization and function of myotonin protein kinase (MtPK), identified as the gene product of myotonic dystrophy, the most prevalent disease with multisystem disorders among muscular dystrophies. To investigate the localization of MtPK, we raised a polyclonal antibody against a synthetic peptide chosen within the deduced sequence of MtPK. This antibody detected both a membrane-bound 70-kd protein and a soluble 55-kd protein on Western blots of human muscles. By using this antibody for immunohistochemical studies of both biopsied human skeletal muscle fibers and mature innervated cultured muscle fibers, we can now demonstrate by confocal laser scanning microscopy that MtPK is localized mainly in the I-band. By immunoelectron microscopy, it was determined that MtPK is a membrane-bound protein localized mainly in the terminal cisternae of the sarcoplasmic reticulum. To our knowledge, this is the first documentation of the ultrastructural localization of MtPK. This finding is quite important for clarifying the pathophysiological basis of myotonic dystrophy, which might be due to a dysregulation of calcium metabolism.     

First mutation (S340X) in choroideremia gene in a Spanish family. Mutations in brief no. 173. Online

A study of choroideremia gene was performed in Spanish families affected with this disorder. One abnormal pattern was detected in exon eight corresponding to a new mutation not described before. The mutation was identified as a nonsense mutation S340X.     

Noval mutation (Y184C) in exon 4 of the beta-sarcoglycan gene identified in a Portuguese patient. Mutations in brief no. 177. Online

We report a novel beta-sarcoglycan gene mutation identified in a 21-year-old Portuguese male with a progressive myopathy of intermediate severity, who had been misdiagnosed as Becker Muscular Dystrophy (BMD) based on clinical observations and muscle immunocytochemical anaylsis with dystrophin antibodies only. Since no detectable deletions or duplications were found in the dystrophin gene, we screened for mutations in the sarcoglycan genes by PCR-SSCP. The patient's sample showed a band of increased mobility in exon 4 of the beta-sarcoglycan gene which, upon sequencing, was found to represent a homozygous A-->G transversion at nucleotide 551, resulting in a tyrosine to cysteine substitution at position 184 (Y184C). Carrier status was ascertained in both parents and a sister. These aberrant conformers were not detected in 85 unrelated control individuals screened by PCR-SSCP analysis. All seven beta-sarcoglycan mutations reported to date are associated with a severe phenotype and occur in exons 3 and 4, which correspond to the immediate extracellular domain of the protein. This region contains five conserved cysteine residues. In our patient, the presence of an extra cysteine residue could interefere with intra- and/or inter-molecular disulphide bond formation. The intermediate phenotype could perhaps result from the assembly of both normal and abnormal complexes, depending on the formation of the disulphide bonds.     

Novel intragenic polymorphisms in the tuberous sclerosis 2 (TSC2) gene. Mutations in brief no. 184. Online

Twenty-three unrelated patients with tuberous sclerosis have been screened for the presence of mutations in six regions of the TSC2 gene. Eight novel intragenic polymorphisms have been found, one in intron 36 and seven in intron 4, with the use of SSCP analysis. Four of these polymorphisms alter the recognition sequence of specific restriction enzymes and can be detected as RFLPs. Study in a random sample of unrelated individuals from Northern Greece, showed that these polymorphisms have mean observed and expected heterozygosity values of 0.2996 and 0.3349, respectively and could be useful for linkage analysis. It is most likely that the wild type alleles from two pairs of these polymorphisms are strongly associated. A 667 bp segment of intron 4 (954 bp) and an additional 75 bp of intron 36 (352bp) were sequenced, thus completing the sequence of both introns.     

A novel mutation (V191G) in a German-British type 1 Gaucher disease patient. Mutations in brief no. 131. Online

Gaucher disease results from mutations in the glucocerebrosidase gene located on human chromosome 1q21. Three clinical forms of Gaucher disease have been described: type 1, nonneuropathic; type 2, acute neuropathic; and type 3, subacute neuropathic. We have identified a novel mutation in a German-British patient with type 1 Gaucher disease which results in V191G of the glucocerebrosidase polypeptide. Because the mutation abolishes a HphI cleavage site, its presence was confirmed by HphI RFLP analysis of PCR-amplified genomic DNA. In the second allele of the patient, the mutation identified was g.5841A G(N370S). Sequence analysis of the remainder of the coding region of the gene as well as the exon-intron boundaries showed identity to normal controls. Because mutation N370S has so far been found only in type 1 Gaucher disease and postulated to result in mild clinical presentation, and since the clinical course of this patient has been relatively mild with minimal skeletal involvement, we speculate that the V191G/N370S genotype may also result in good prognosis.     

Iraqi-Jewish kindreds with optic atrophy plus (3-methylglutaconic aciduria type 3) demonstrate linkage disequilibrium with the CTG repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene

Iraqi-Jewish optic atrophy plus is an autosomal recessive condition characterized by infantile optic atrophy, an early onset movement disorder, and 3-methylglutaconic aciduria. Other features include spastic paraplegia, mild ataxia, mild cognitive deficiency and dysarthria. This disorder was identified in inbred Iraqi-Jewish kindreds in which relationships between most of the affected individuals were unknown. In this study we identify linkage to chromosome 19q13.2-q13.3 by using a DNA pooling strategy to perform a genome wide screen followed by a high density search for shared segments among affected individuals in candidate regions identified in the initial genome wide screen. A significantly high positive lod score of 6.14 at zero recombination was obtained for the CTG repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene. The existence of multiple recombinant individuals indicates the disease interval can be further narrowed with additional markers. Linkage disequilibrium was seen in six polymorphic markers across a 1 Mb interval. This region is well characterized and contains several candidate genes.      

Newly recognized exons induced by a splicing abnormality from an intronic mutation of the dystrophin gene resulting in Duchenne muscular dystrophy. Mutations in brief no. 213. Online

A boy with the clinical phenotype of Duchenne muscular dystrophy had no detectable deletion or duplication in the dystrophin gene by the routine multiplex PCR method. In mRNA extracted from his muscle biopsy, newly recognized extra-exons of 172 bp and 202 bp were present between exon 25 and 26 suggesting a splicing abnormality. Genomic DNA of the intron 25 including the above insertions were amplified and sequenced. There was one nucleotide substitution of A-to-G at 2 Kb downstream from the 5' end of intron 25 which formed consensus dinucleotide 'GT' motif for 5' splice site resulting in aberrant splicing. This is the first patient who had a mutation at the central part of an intron of the dystrophin gene instead of at the exon-intron border.     

Mutations of the human P gene associated with Type II oculocutaneous albinism (OCA2). Mutations in brief no. 205. Online

Mutations in the human P gene lead to oculocutaneous albinism type 2 (OCA2, MIM #203200), the most common type of albinism in humans. The P gene encodes a 110 kDa protein that is associated with melonosomal membranes and contains 12 potential membrane spanning domains. The specific function of the P protein is currently unknown. We report 7 new mutations in the P gene associated with OCA2. This includes 6 missense mutations (S86R, C112F, A368V, T592I, A724P and A787V) and one frameshift mutation (1047del7). We also report 8 polymorphisms including one amino acid substitution, D/A257. We and others have found many polymorphisms of the P gene in the coding region, several of which result in amino acid substitutions, making molecular diagnosis problematic. In contrast to this is the tyrosinase gene associated with OCA1, with a limited number of polymorphic variations in the coding region. There is also no apparent clustering of P gene missense mutations in contrast to the clustering observed by the tyrosinase gene missense mutations that define functional domains of the protein. Further mutational analysis is needed to help define the critical functional domains of the P protein and to allow a definitive diagnosis of OCA2.     

Mutations of the human tyrosinase gene associated with tyrosinase related oculocutaneous albinism (OCA1). Mutations in brief no. 204. Online

Mutations in the human tyrosinase gene produce tyrosinase-related oculocutaneous albinism (OCA1, MIM #203100). Tyrosinase is a copper containing enzyme and is responsible for catalyzing the rate limiting step in melanin biosynthesis, the hydroxylation of tyrosine to dopaquinone. We report 13 new mutations in the tyrosinase gene associated with OCA1A (without pigment) and OCA1B (with pigment) including 9 missense mutations (H19Q, R521, R77C, G97R, C289R, L312V, P313R, F340L and H404P), two nonsense mutations (W80X and R116X) and two frameshift mutations (53delG and 223 delG). Our previous work has defined clusters of missense mutations that appear to represent functional domains of the enzyme, and three of the missense mutations fall into these clusters including two (F340L and H404P) that flank the copper B bindng site and the missense mutation R52I that is located in the amino terminal end cluster of the protein. The G97R missense mutation is the first identified within the epidermal growth factor (EGF)-like sequence and the H19Q missense mutation alters the cleavage site of the signal peptide sequence. Mutational analysis can provide a definitive diagnosis of the type of OCA as well as help structure/function analysis.     

French myotonic dystrophy families show expansion of a CTG repeat in complete linkage disequilibrium with an intragenic 1 kb insertion.

The molecular basis of myotonic dystrophy (DM) has been characterised. All DM mutations characterised to date appear as an unstable elongation of a fragment containing a tandem repeat of a CTG motif, which can be visualised in both EcoRI and BamHI digests. It has been shown that the fragment is polymorphic in the normal population. Another 1 kb insertion/deletion polymorphism located near the unstable CTG repeat region has been identified. The 1 kb insertion allele is present in all DM patients. These different polymorphic systems can be distinguished using cDNA25 and BamHI, because this enzyme cuts between the site of the 1 kb insertion and the CTG repeat. We thus haplotyped DM patients from 72 French families and clearly showed that all chromosomes (100%) with the DM mutation carried the 1 kb insertion as well. In addition to this association, we detected significant linkage disequilibrium between the DM locus and D19S63 for which allelic frequencies were different from other European populations. Our results in the French DM population are thus in agreement with the hypothesis that the CTG expansion occurred on one or a few ancestral chromosomes carrying the large 1 kb insertion allele.     

A genetic polymorphism in the human HOXB1 homeobox gene implying a 9bp tandem repeat in the amino-terminal coding region. Mutations in brief no. 200. Online

In man there are 39 homeobox genes of the HOX family in four loci, HOXA, HOXB, HOXC and HOXD on chromosomes 7, 17, 12, and 2. We discovered the existence of two major alleles, termed a and b, of gene HOXB1. They differ at a specific position in the 5' portion of the coding region. Sequencing the two alleles revealed that the allele HOXB1A, contains two copies of the 9bp sequence 5'ACAGCGCCC3', starting at position 65 of the coding region, whereas the allele HOXB1b contains three copies of this sequence (Fig. 1). As a consequence, the allele HOXB1b encodes a homeoprotein containing two copies of the tripeptide HisSerAla, starting at amino acid residue 25, which is present in only one copy in allele HOXB1a. We analyzed 250 individuals and found that the allelic frequencies of HOXB1a and HOXB1b were 78.8% and 21.2%. The murine homologue contains only one copy of the 9bp repeat (Fig. 1). 7 mouse strains, namely 129, BALB/c, C57BL/6, C57BL/10, CAST/Ei, C3H and SPRET/Ei, are homozygous for this allele. The allele present in gibbon and rhesus monkey appears to be identical to the human HOXB1b (Fig. 1).