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Neurogenetics. 2004 February; 5(1): 27–34.
Published online 2003 December 2. doi: 10.1007/s10048-003-0165-9.

New FKRP mutations causing congenital muscular dystrophy associated with mental retardation and central nervous system abnormalities. Identification of a founder mutation in Tunisian families

Nacim Louhichi,1 Chahnez Triki,2 Susana Quijano-Roy,3 Pascale Richard,4 Samira Makri,5 Mériem Méziou,2 Brigitte Estournet,3 Slah Mrad,3 Norma B. Romero,6 Hammadi Ayadi,1 Pascale Guicheney,6 and Faiza Fakhfakh1*

1Laboratoire de Génétique Moléculaire Humaine Faculté de Médecine de Sfax, 3029, Sfax,TN
2Service de neurologie CHU Habib Bourguiba, 3029, Sfax,TN
3Service de Pédiatrie, Rééducation et Réanimation Neurorespiratoire AP-HP, Hôpital Raymond Poincaré, 92380 Garches,FR
4Unité Fonctionnelle de Cardiogénétique et Myogénétique Hôpital de La Pitié-Salpêtrière, Paris,FR
5Etablissement Hospitalier Spécialisé Ali Ait Idir Etablissement Hospitalier Spécialisé Ali Ait Idir, 16000 Hahad, Alger,DZ
6Physiopathologie et thérapie du muscle strié INSERM : U582, IFR14, Université Pierre et Marie Curie - Paris VI, Gh Pitie-Salpetriere 47, Boulevard de L'Hopital 75651 PARIS CEDEX 13,FR
Abstract

The congenital muscular dystrophies (CMD) constitute a clinically and genetically heterogeneous group of autosomal recessive myopathies. Patients show congenital hypotonia, muscle weakness and dystrophic changes on muscle biopsy. Mutations in four genes (FKT1, POMGnT1, POMT1, FKRP) encoding for putative glycosyltransferases have been identified in a subset of patients which is characterized by a deficient glycosylation of α-dystroglycan on muscle biopsy. FKRP mutations account for a broad spectrum of patients with muscular dystrophy, from a severe congenital form with or without mental retardation (MDC1C) to a much milder limb-girdle muscular dystrophy (LGMD2I). We identified two novel homozygous missense FKRP mutations, one, A455D, in six unrelated Tunisian patients and the other, V405L, in an Algerian boy. The patients, between the age of 3 and 12 years, presented with a severe form of MDC1C with calf hypertrophy, high serum creatine kinase levels. None had never walked. Two had cardiac dysfunction and one strabismus. They all had mental retardation, microcephaly, cerebellar cysts, and hypoplasia of the vermis. White matter abnormalities were found in five of them, mostly when cranial MRI was performed at a young age. These abnormalities were shown to regress in one case, as has been observed in FCMD patients. Identification of a new microsatellite close to FKRP allowed us to confirm the founder origin of the Tunisian mutation. These results suggest strongly that particular FKRP mutations in the homozygous state induce structural and clinical neurological lesions in addition to muscular dystrophy and relate MDC1C to other CMDs with abnormal protein glycosylation and disordered brain function.

MeSH keywords: Abnormalities, Multiple, genetics, Cerebellum, abnormalities, Child, Child, Preschool, Family Health, Female, Founder Effect, Glycosylation, Haplotypes, Homozygote, Humans, Magnetic Resonance Imaging, Male, Mental Retardation, complications, genetics, Muscular Dystrophies, complications, congenital, genetics, Pedigree, Proteins, genetics, metabolism, Tunisia

Author keywords: Congenital muscular dystrophy, FKRP gene, founder-haplotype, mental retardation, cerebellar cysts

INTRODUCTION

Congenital muscular dystrophies (CMD) constitute a heterogeneous family of autosomal recessively inherited diseases, presenting at birth or within the first few weeks of life with hypotonia, delayed motor development and dystrophic changes on skeletal-muscle biopsy (1). Clinical heterogeneity is evidenced by different patterns of motor involvement, variable serum creatine kinase (CK) levels and the presence or not of mental retardation and structural defects of central nervous system (CNS). At least one third of patients in western countries have a primary deficiency in the laminin α2 chain (merosin) resulting from mutations in the LAMA2 gene (2, 3, 4). This type of CMD, usually referred as merosin-deficient CMD, has now been classified as MDC1A and is always associated with diffuse white matter abnormalities, and occasionally cerebellar hypoplasia or abnormal lesions of the cerebral gyri (5, 6, 7). Epileptic seizures have been reported in a subset of these patients. A secondary deficiency of laminin α2 chain and of α-dystroglycan is found in some CMD forms including muscle-eye-brain disease (MEB), Walker-Warburg syndrome (WWS), Fukuyama CMD (FCMD), and MDC1C, mapped to chromosomes 1p32, 9q34, 9q31 and 19q13, respectively (8, 9, 10, 11). These CMDs are caused by defects in genes encoding for putative glycosyltransferases: POMGnT1 gene coding for an O-mannose glycosyltransferase (MEB) (8), POMT1 gene coding for O-mannosyltransferasel (WWS) (9), FKT1 gene encoding a protein of unknown function called fukutin (FCMD) (10), and FKRP, a homologue of the FKT1 gene (MDC1C) (11). Clinically, the first three types show severe symptoms and structural central nervous system involvement. MDC1C is characterized by marked elevation of serum creatine kinase (CK), early onset hypotonia, delayed or arrested motor development, muscle hypertrophy, and variable cardiomyopathy. However, no major CNS abnormalities have been described in initial reports even in the most severe forms (11, 12). Very recently, two homozygous FKRP mutations associated with mental retardation and cerebellar cysts have been identified in two Turkish patients (13). They were isolated cases and, therefore, it was not possible to conclude if particular FKRP gene mutations may constitute a subset of patients with a particular phenotype characterized by severe neurological involvement. To address this issue, we report two new FKRP mutations associated with CMD and mental retardation, one in a series of six unrelated Tunisian families and another in an Algerian case. All the cases had mental retardation and neuro-imaging showed a variable combination of CNS abnormalities including cerebellar cysts or atrophy with or without white matter lesions. These results suggest strongly that certain FKRP mutations induce structural and clinical neurological abnormalities in a subset of patients with MDC1C. It may help in better understanding of this entity and relates this type of CMD to others with abnormal protein glycosylation and structural CNS abnormalities. In addition, identification of a new microsatellite close to the FKRP gene was used to investigate the founder origin of the Tunisian mutation.

PATIENTS AND METHODS
Linkage and haplotype analysis
Genetic analysis was performed on genomic DNA extracted from blood leukocytes using a standard phenol-chloroform procedure (14). Microsatellites spanning the FKRP locus (D19S606, D19S412, D19S219 and fkrp 52) were studied. PCR amplification was carried out in 50 μl with 60 ng of genomic DNA, 1 μM of each primer, 125 μM dNTPs, 1.5 mM MgCl2, 5 mM KCl, 10 mM Tris-HCl, pH 8.8 and 1U of Taq DNA polymerase. PCR products were analyzed on 6 % denaturing polyacrylamide gels, transferred on to N+ Hybond membrane (Amersham Pharmacia Biotech) and hybridized with a poly AC probe labeled with α32P. The following primers were designed to amplify the new microsatellite, fkrp 52 (ID = ss12568442 in SNP NCBI database): 5′-TCTCCAAAAAACAACAACAAC-3′ and 5′-CTAGTGTTCTGGGACCTTT-3′. The sequence of the amplified fragment is available in the Third Party Annotation Section of the DDBJ/EMBL/Genbank databases under the accession number TPA: BK001438.
FKRP mutational analysis
A 1.8kb fragment containing the FKRP coding sequence of exon 4 and its bordering intronic sequences was amplified using 5 primer sets (Table 1). The overlapping PCR products were purified (Qiagen), and used for direct sequencing on both strands. Sequencing reactions were carried out using an ABI Prism Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems). The 1364C>A mutation induces the loss of a NaeI restriction site. NaeI restriction pattern of the FKRP 4–5 fragment (421bp) was used to confirm the presence of the mutation in patients’s relatives and to screen 100 unrelated Tunisian, healthy individuals. Digestion of PCR products was performed according to the manufacturer’s instructions (New England Biolabs), followed by separation on 2% Nusieve gels. The 1213>T mutation identified in the FKRP 4–5 fragment was searched by direct sequencing in 50 Algerian and 100 French healthy control individuals.
RESULTS
Clinical and complementary findings
The main clinical features of the Tunisian (Patients 1 to 6) and Algerian patients are summarized in Table 2.
Patients 1, 2 and 3. These patients have previously been described (15); they presented a severe form of CMD with mental retardation and cerebellar cysts. The state of these patients worsened progressively. For Patient 1, brain MRI performed at ages of 3 and 6 showed a significant reduction of the white matter abnormalities with age (Figure 1). In contrast, the hypoplasia of the pons and cerebellar cysts remained unchanged.
Patient 4. This patient was a 5 year-old girl. She had a severe form of CMD with mental retardation. EMG was myopathic and muscle biopsy showed severe dystrophic features. Cranial MRI at age 5 revealed only cerebellar cysts without any white matter or other cerebellar abnormality (Figure 2). Echocardiogram showed left ventricular hypertrophy.
Patient 5. This patient was a 6 year-old girl. She presented since birth a severe form of CMD with axial and facial weakness and had mental retardation. The ophthalmological examination was normal. Brain CT scan had been performed at the age of three years and showed white matter abnormalities (not shown).
Patient 6. Patient 6 was a twelve year-old mentally retarded girl who had an older brother with congenital muscle dystrophy associated with encephalopathy who had died at the age of three due to a pneumopathie. She was the oldest child of this series and was the most severely affected patient. She never walked. She showed a severe facial weakness, progressive weakness, macroglossia, limited ocular excursion and scoliosis which was treated surgically. She was mechanically ventilated by age 10 because of severe restrictive respiratory insufficiency. At 12 years she was only able to perform minimal movements of the fingers which were contracted in flexion (Figure 2) and a mild cardiac involvement was detected, with a reduced shortening fraction and a normal ejection fraction. She did not speak a word before 5 years of age. At 12 years, she was able to say only single simple words of two or three syllables (Verbal IQ: 50) and her brain MRI showed cerebral atrophy, very white matter abnormalities and hypoplasia of cerebellar vermis and cerebellar cysts (Figure 2). White matter abnormal signal was very diffuse in early life and regressed progressively.
Patient 7. This patient was a 3 year-old mentally retarded boy. He presented since birth with severe form of CMD. EMG showed myopathic features and the muscle biopsy of the tibialis muscle showed severe dystrophic features. ECG, EEG and ophthalmological examination were normal. Brain MRI revealed cerebellar cysts, megacisterna with hypoplasia of the vermis and an abnormally high periventricular intensity on T2 weighed images. At the age of three years, he was microcephalic and could say only a few words.
Immunocytochemistry
All the patients showed histological changes characteristic of muscular dystrophy, and reduced immunohistochemical staining of laminin α2 chain using antibodies against the 80 and the 300 kDa fragments; α-dystroglycan expression was found to be markedly reduced in the only two samples available (data not shown).
Mutation screening of the FKRP gene
After exclusion of the CMD loci (FCMD, MEB, MDC1A and MDC1B) in Families 1–7 (data not shown), all patients were found to be homozygous for the markers surrounding the FKRP locus (D19S219, D19S412, D19S606) on chromosome 19q13.3, suggesting homozygous mutations in FKRP (Figure 5, data not shown for Family 7). Sequencing of the FKRP coding region revealed the same homozygous transversion, 1364C>A, in the six Tunisian patients inducing the change of alanine 455 to a negatively charged aspartic acid (A455D) (Figure 3). We took advantage of the loss of a NaeI restriction site induced by this mutation to rule it out as a non-pathogenic polymorphism. This variant was not found in 200 control chromosomes. Using NaeI digestion and direct sequencing, the mutation was found to segregate with the disease in an autosomal recessive fashion in the six consanguineous families (Figures 4 and 5). A second homozygous transversion 1213G>T was identified in the Algerian patient changing valine 405 to a leucine (V405L). This change was not found by direct sequencing in 50 Algerian and 100 French healthy control individuals.

Since the 6 families originated from Tunisia, we searched for a founder origin of the mutation. The known markers flanking FKRP, D19S219, D19S412 and D19S606, were physically mapped according to the Human Genome Browser and Ensembl Genome Server maps (http://genome.cse.ucsc.edu/goldenPath/hgTracks.html and http://www.ensembl.org). They are located at 1255 kb and 238 kb proximal, and 722 kb distal to FKRP, respectively. Among the families tested, Families 1 to 4 belonging to the same region in the South of Tunisia shared the same alleles (Figure 5) while the two others did not. We searched for new (CA)n microsatellite markers closer to FKRP. One was identified, fkrp 52, with alleles varying from 92 to 122 bp, 42 kb proximal to FKRP. A rare 120 bp allele containing 26 CA repeats was shared by the 6 families suggesting that the mutation has been transmitted by a common ancestor (Figure 5).

DISCUSSION

In this study, we report two new homozygous missense FKRP mutations in 7 patients affected with MDC1C associated with mental retardation and CNS abnormalities. Six of them originating from Tunisia share the same mutation, which strongly suggests that this particular FKRP mutation in the homozygous state invariably leads to the CNS involvement.

Mutations in the FKRP gene previously reported account for a broad spectrum of patients with muscular dystrophy, from a severe congenital form (MDC1C) (11) to a much milder limb-girdle muscular dystrophy (LGMD2I) (16). There exist intermediate phenotypes which look like Duchenne muscular dystrophy. The intermediate and congenital forms may share an identical end-stage picture characterized by a paralytic atrophic-hypertrophic phenotype including extreme diffuse weakness, tongue hypertrophy and need of continuous mechanical ventilation (11, 12). Genotype-phenotype correlation has been evidenced by the identification of “mild” and “severe” mutations in cases with LGMD2I and MDC1C respectively, and which lead to an intermediate phenotype when combined (17, 18, 19). In spite of differences in the degree of severity, a common phenotypical pattern including some features such as high CK levels, muscle hypertrophy, a progressive course and reduced expression of laminin α2 chain and α-dystroglycan in muscle characterizes this entity.

The patients of our series presented with a typical MDC1C phenotype, but in addition they had severe psycho-motor retardation and developed a microcephaly which was not observed at birth. They showed late and unsteady head control, inability to stand or walk, and marked mental retardation. Very high CK levels were constant, and calf hypertrophy was noted in four of the patients. The oldest patient developed at the end of the first decade the typical paralytic atrophic-hypertrophic FKRP phenotype. This was not observed in the other six patients probably because they were too young to manifest this progression which is not usually observed in the first years of life (12). Regarding neuroradiological findings, all the patients in our series showed white matter changes and/or cerebellar structural abnormalities. We have previously reported the presence of minor signs of central involvement such as speech delay, brain atrophy and mild white matter lesions in some MDC1C patients, but such findings were thought to be non-specific or secondary to hypoxic complications related to the respiratory insufficiency in advanced stages of the disease (12). Therefore, MDC1C was initially classified as a CMD without neurological involvement, in contradistinction to the CMD-syndromes with structural brain abnormalities such as MEB, FCMD and WWS. Brain abnormalities such as agyria, pachygyria or polymicrogyria, hypomyelination of the white matter, cerebellar cysts or ponto-cerebellar hypoplasia are frequent findings in these three conditions (8, 9, 10). In our series, brain MRI was available for six of the seven patients and revealed cerebellar cysts in all of them, and hypoplasia of the vermis in five. MRI has not been performed in Patient 5 and, therefore, we cannot exclude the presence of posterior fossa abnormalities in this patient. Our patients did not show any gyral malformation in contrast to what may be observed in MEB, FCMD or WWS. White matter abnormalities were observed in five of our patients, with significant regression in one of them, detected by serial MRIs at 3 and 6 years of age. Such transient infra-myelination has also been described in MEB and FCMD (19, 20) while in primary laminin α2 chain deficient CMD, brain white matter abnormalities do not regress with time. It is also noteworthy that one patient presented with strabismus and another with ophthalmoplegia in the second decade of life.

Very recently, different FKRP homozygote mutations were identified in three Turkish patients, two with mental retardation, white matter changes and cerebellar cysts (13), and another with a typical Walker-Warburg syndrome (Beltran-Valero de Bernabé et al, 2003, personal communication). This finding raised the question of the role of specific homozygous FKRP mutations in the development of neurological disturbances. Our study revealing two new homozygous missense FKRP mutations, A455D and V405L, in seven additional patients of similar phenotype clearly supports this hypothesis. Fukutin related protein has been shown to be localized in the Golgi apparatus (21). It is involved in the glycosylation processing of α-dystroglycan, a heavily glycosylated membrane protein that forms a link between the actin associated cytoskeleton and the extracellular matrix via the laminin α2 chain (22). Hypoglycosylation of α-dystroglycan abolishes binding not only to laminin, but also to neurexin and agrin, and such abnormal interactions underlie the pathogenic mechanism of muscular dystrophy with brain abnormalities (23, 24). The mutations identified in our patients, V405L and A455D, are localized in the C-terminal domain (catalytic domain) of the fukutin related protein, where other severe MDC1C mutations (P448L, Y465S) without CNS involvement have also been identified. These different mutations may affect in various degrees the conformation or the catalytic activity of the protein and/or recognition sites for α-dystroglycan and other potential substrates (16). In contrast, the two mutations reported by Topaloglu et al, P315T and S221A, which cause a similar phenotype with CNS involvement are located outside the catalytic domain in the stem region. This region is believed to be involved in protein-protein interaction (13).

In summary, the report describes two new missense mutations in the FKRP gene in seven children with MDC1C, mental retardation and central nervous system abnormalities. While brain white matter changes may regress with time, structural cerebellar abnormalities were present early in life and did not seem to progress. Our results support the existence of particular homozygous FKRP mutations associated with severe neurological involvement, and relate MDC1C to other congenital muscular dystrophies with abnormal protein glycosylation and affecting brain function.

 
Acknowledgments
 
Figures and Tables
Figure 1Figure 1
T2-Weigted axial MRI of the brain of Patient 1 shows abnormal high white matter signal intensities at age of 3 (a) and regression of these abnormalities on follow-up examination at the age of 6 (b).
Figure 2Figure 2
A- Transverse T2-Weighted MRI image from Patient 4 showing cerebellar cysts.
Figure 3Figure 3
Sequence chromatograms from normal individual (wild type, WT) and affected individuals (P) are shown together with the expected amino-acid changes. Nucleotide variations are indicated by an arrow. A: 1364C>A inducing the A455D mutation in Patient 1 (P1), (more ...)
Figure 4Figure 4
Restriction enzyme analysis showing the inheritance of the 1364C>A mutation in family 4. A 421bp PCR fragment was digested with Nae I. Normal control DNA is cut into two fragments of 272 bp and 149 bp, whereas products containing the mutation remained (more ...)
Figure 5Figure 5
The pedigrees of the 6 Tunisian families showing the inheritance of the 1364C>A mutation. The haplotypes containing the mutation are boxed. We note the same founder haplotype for Families 1, 2, 3 and 4. Families 5 and 6 had the same mutation but not the (more ...)
Table 1Table 1
PCR primers used to amplify the coding sequence of the FKRP gene located within exon 4
Table 2Table 2
Summary of the clinical features of the seven patients (CK creatine kinase, ND no data)