André Reis

NIBRIN, A NOVEL DNA DOUBLE-STRAND BREAK REPAIR PROTEIN, IS MUTATED IN NIJMEGEN BREAKAGE SYNDROME

Nijmegen breakage syndrome (NBS) is an autosomal recessive chromosomal instability syndrome characterized by microcephaly, growth retardation, immunodeficiency, and cancer predisposition. Cells from NBS patients are hypersensitive to ionizing radiation with cytogenetic features indistinguishable from ataxia telangiectasia. We describe the positional cloning of a gene encoding a novel protein, nibrin. It contains two modules found in cell cycle checkpoint proteins, a forkhead-associated domain adjacent to a breast cancer carboxy-terminal domain. A truncating 5 bp deletion was identified in the majority of NBS patients, carrying a conserved marker haplotype. Five further truncating mutations were identified in patients with other distinct haplotypes. The domains found in nibrin and the NBS phenotype suggest that this disorder is caused by defective responses to DNA double-strand breaks.

Mutations in the gene encoding the lamin B receptor produce an altered nuclear morphology in granulocytes (Pelger-Huet anomaly).

Pelger–Huët anomaly (PHA; OMIM *169400) is an autosomal dominant disorder characterized by abnormal nuclear shape and chromatin organization in blood granulocytes. Affected individuals show hypolobulated neutrophil nuclei with coarse chromatin. Presumed homozygous individuals have ovoid neutrophil nuclei, as well as varying degrees of developmental delay, epilepsy and skeletal abnormalities. Homozygous offspring in an extinct rabbit lineage showed severe chondrodystrophy, developmental anomalies and increased pre- and postnatal mortality. Here we show, by carrying out a genome-wide linkage scan, that PHA is linked to chromosome 1q41–43. We identified four splice-site, two frameshift and two nonsense mutations in LBR, encoding the lamin B receptor. The lamin B receptor (LBR), a member of the sterol reductase family, is evolutionarily conserved and integral to the inner nuclear membrane; it targets heterochromatin and lamins to the nuclear membrane. Lymphoblastoid cells from heterozygous individuals affected with PHA show reduced expression of the lamin B receptor, and cells homozygous with respect to PHA contain only trace amounts of it. We found that expression of the lamin B receptor affects neutrophil nuclear shape and chromatin distribution in a dose-dependent manner. Our findings have implications for understanding nuclear envelope–heterochromatin interactions, the pathogenesis of Pelger-like conditions in leukemia, infection and toxic drug reactions, and the evolution of neutrophil nuclear shape.

A major susceptibility locus for atopic dermatitis maps to chromosome 3q21

Atopic dermatitis (eczema) is a chronic inflammatory skin disease with onset mainly in early childhood. It is commonly the initial clinical manifestation of allergic disease, often preceding the onset of respiratory allergies. Along with asthma and allergic rhinitis, atopic dermatitis is an important manifestation of atopy that is characterized by the formation of allergy antibodies (IgE) to environmental allergens. In the developed countries, the prevalence of atopic dermatitis is approximately 15%, with a steady increase over the past decades. Genetic and environmental factors interact to determine disease susceptibility and expression, and twin studies indicate that the genetic contribution is substantial. To identify susceptibility loci for atopic dermatitis, we ascertained 199 families with at least two affected siblings based on established diagnostic criteria. A genome-wide linkage study revealed highly significant evidence for linkage on chromosome 3q21 (Zall=4.31, P= 8.42×10−6). Moreover, this locus provided significant evidence for linkage of allergic sensitization under the assumption of paternal imprinting (hlod=3.71, α=44%), further supporting the presence of an atopy gene in this region. Our findings indicate that distinct genetic factors contribute to susceptibility to atopic dermatitis and that the study of this disease opens new avenues to dissect the genetics of atopy.

Dysferlin deletion in SJL mice (SJL-Dysf) defines a natural model for limb girdle muscular dystrophy 2B.

Dysferlin deletion in SJL mice (SJL- Dysf ) defines a natural model for limb girdle muscular dystrophy 2B

Splitting schizophrenia: periodic catatonia-susceptibility locus on chromosome 15q15.

The nature of subtypes in schizophrenia and the meaning of heterogeneity in schizophrenia have been considered a principal controversy in psychiatric research. We addressed these issues in periodic catatonia, a clinical entity derived from Leonhards classification of schizophrenias, in a genomewide linkage scan. Periodic catatonia is characterized by qualitative psychomotor disturbances during acute psychotic outbursts and by long-term outcome. On the basis of our previous findings of a lifetime morbidity risk of 26.9% of periodic catatonia in first-degree relatives, we conducted a genome scan in 12 multiplex pedigrees with 135 individuals, using 356 markers with an average spacing of 11 cM. In nonparametric multipoint linkage analyses (by GENEHUNTER-PLUS), significant evidence for linkage was obtained on chromosome 15q15 (P = 2.6 x 10(-5); nonparametric LOD score [LOD*] 3.57). A further locus on chromosome 22q13 with suggestive evidence for linkage (P = 1.8 x 10(-3); LOD* 1.85) was detected, which indicated genetic heterogeneity. Parametric linkage analysis under an autosomal dominant model (affecteds-only analysis) provided independent confirmation of nonparametric linkage results, with maximum LOD scores 2.75 (recombination fraction [theta].04; two-point analysis) and 2.89 (theta =.029; four-point analysis), at the chromosome 15q candidate region. Splitting the complex group of schizophrenias on the basis of clinical observation and genetic analysis, we identified periodic catatonia as a valid nosological entity. Our findings provide evidence that periodic catatonia is associated with a major disease locus, which maps to chromosome 15q15.

Diaphragmatic Spinal Muscular Atrophy with Respiratory Distress Is Heterogeneous, and One Form Is Linked to Chromosome 11q13-q21

To the Editor: Diaphragmatic spinal muscular atrophy (SMA) has been delineated as a variant of infantile SMA (SMA1 [MIM 253300]) (Mellins et al. 1974; Bertini et al. 1989). The most prominent symptoms are severe respiratory distress resulting from diaphragmatic paralysis with eventration shown on chest x-ray and predominant involvement of the upper limbs and distal muscles. In contrast to classic SMA1, in diaphragmatic SMA the upper spinal cord is more severely affected than the lower section. The pmn mouse presents with progressive motor neuronopathy and a disease that closely resembles diaphragmatic SMA (Schmalbruch et al. 1991). The pmn locus has been mapped to murine chromosome 13 (Brunialti et al. 1995). Here we report on nine patients from three families with diaphragmatic SMA following autosomal recessive inheritance. The diagnosis of diaphragmatic SMA was made on the basis of clinical criteria (Rudnik-Schoneborn et al. 1996). Family 1 is of Lebanese origin; family 2, German origin; and family 3, Italian origin. We obtained DNA samples from these families after receiving informed consent, in accordance with the Declaration of Helsinki. In family 1 (fig. 1A), the parents are first cousins. The first affected son died, at the age of 10 wk, of suspected sudden infant death syndrome (SIDS). One daughter presented, at the age of 6 wk, with feeding difficulties and progressive respiratory distress. Chest x-ray showed eventration of the diaphragm. Mechanical ventilation was initiated at the age of 8 wk. She developed progressive muscular atrophy with complete paralysis of the upper and lower limbs and mild contractures of the knee and ankle joints. Three other children, nonidentical twin daughters and the youngest daughter, died of respiratory failure—the twins at the age of 8 and 9 wk and the youngest daughter at the age of 8 wk. Autopsy specimens were taken from gastrocnemius muscle in both twins and from the upper spinal cord in one twin. Skeletal-muscle histology revealed neurogenic atrophy without signs of reinnervation. Ultrastructurally, the motor end plates lacked nerve terminals and showed postsynaptic degenerative changes characterized by deep invaginations. The diameter of anterior spinal roots was reduced in the upper spinal cord. The remaining motor neurons showed chromatolysis. These findings offer two different pathophysiological concepts: (1) degeneration of the anterior horn cells of the spinal cord with neurogenic muscular atrophy suggests dying-forward atrophy, and (2) presynaptic and postsynaptic signs of motor end-plate degeneration suggest dying-back atrophy. In family 2 (fig. 1B), the first child had severe muscular hypotonia and died, at the age of 9 wk, of cardiorespiratory failure. The third child has been mechanically ventilated since the age of 3 mo. In family 3 (fig. 1C), which has been reported in detail elsewhere (Novelli et al. 1995), the gene locus for SMA1, on chromosome 5q, has been excluded. Both affected sibs presented with respiratory insufficiency right after birth and with the typical signs of diaphragmatic SMA. Figure 1 Haplotypes in families with diaphragmatic SMA subtypes. A, Family 1 (Lebanese origin): age at onset, 6–10 wk. B, Family 2 (German origin): age at onset, 9–12 wk. C, Family 3 (Italian origin): onset at birth. Haplotype analysis indicated ... First, we confirmed that, in families 1 and 2, there is no linkage of the trait to markers of the SMA locus on 5q11.2-q13.3, as there is in family 3. Second, the orthologous regions corresponding to the murine pmn gene region on human chromosomes 1q and 7p were excluded as gene loci responsible for the disease (Grohmann et al. 1998). To locate the gene locus for diaphragmatic SMA, a whole-genome scan was undertaken in family 1. Microsatellite analysis was performed, by standard semiautomated methods, by an ABI 377-Sequencer, and the results were processed by GENESCAN software, as described elsewhere (Saar et al. 1997). The whole-genome linkage scan was performed with the use of 340 polymorphic fluorescence–labeled markers spaced at ∼10-cM intervals throughout the autosomal part of the genome. Subsequent fine mapping was performed with eight additional microsatellite markers. Markers were chosen from the Genethon final linkage map. Two-point parametric linkage analyses were performed with the LINKAGE package, version 5.2 (Lathrop and Lalouel 1984), under the following assumptions: a regular, fully penetrant autosomal recessive trait locus with a disease-allele frequency of .002 and no phenocopy rate, codominant marker loci with uniformly distributed allele frequencies, and standard recombination rates. Multipoint analysis was performed with the GENEHUNTER program, version 1.3 (Kruglyak et al. 1996). Genomewide linkage scanning of family 1 revealed linkage of diaphragmatic SMA only to markers on chromosome 11q13-q21. In the following, we name this subtype of diaphragmatic SMA “spinal muscular atrophy with respiratory distress” (SMARD). For the markers D11S1296, D11S4095, D11S901, D11S1358, and D11S1757, a maximum two-point LOD score of 3.16 at recombination fraction (θ) 0 was obtained. The two-point LOD scores for 13 markers on chromosome 11q are summarized in table 1. Haplotype analysis revealed a recombination event in individual 2.4 that placed the disease locus distal to marker D11S1883 (fig. 1A). The crossing-over in individual 2.1 placed the disease locus proximal to marker D11S917. Consistent with parental consanguinity, all affected siblings from family 1 were autozygous for all markers within the cosegregating segment. Multipoint linkage analysis with the use of 13 markers yielded a maximum LOD score of 3.86, which clearly places the disease locus between D11S1883 and D11S917 (Genethon map positions 68.5 cM and 100.9 cM). Table 1 LOD-Score Values at Standard Recombination Rates for Markers on Chromosome 11q in Lebanese Family 1 In family 2, the two affected sibs shared two identical parental haplotypes in the SMARD cosegregating segment on 11q13-q21, a finding that supports the assignment of the SMARD locus to this region (fig. 1B). In family 3, haplotype analysis was inconsistent with linkage to the markers tested (fig. 1C). Thus, this locus was excluded as being responsible for the disease in this family. Our finding that diaphragmatic SMA with onset at age 6–12 wk is linked to chromosome 11q markers in two apparently unrelated families from different countries (families 1 and 2) but that diaphragmatic SMA with onset at birth does not show such linkage (family 3) suggests that diaphragmatic SMA is both clinically and genetically heterogeneous. The prevalence of diaphragmatic SMA is unknown. However, in a series of >200 patients with early-onset SMA, ∼1% presented with diaphragmatic SMA and did not have a deletion of the survival motor-neuron gene (SMN) on chromosome 5q (Rudnik-Schoneborn et al. 1996). Considering the case history of the affected son from family 1 who had suspected SIDS, we presume that some of those infants with SIDS may possibly have been misdiagnosed. We are currently looking for further patients with SMARD, to refine the large cosegregating region on chromosome 11q.

Localisation of a gene for Papillon-Lefèvre syndrome to chromosome 11q14-q21 by homozygosity mapping.

Abstract Papillon-Lefèvre syndrome is an autosomal recessively inherited palmoplantar keratoderma of unknown aetiology associated with severe periodontitis leading to premature loss of dentition. Three consanguineous families, two of Turkish and one of German origin, and three multiplex families, one of Ethiopian and two of German origin, with 11 affected and 6 unaffected siblings in all were studied. A targeted genome search was initially attempted to several candidate gene regions but failed to demonstrate linkage. Therefore a genome-wide linkage scan using a combination of homozygosity mapping and traditional linkage analysis was undertaken. Linkage was obtained with marker D11S937 with a maximum two-point lod score of Zmax = 6.1 at recombination fraction θ = 0.00 on chromosome 11q14–q21 near the metalloproteinase gene cluster. Multipoint likelihood calculations gave a maximum lod score of 7.35 between D11S901 and D11S1358. A 9.2-cM region homozygous by descent in the affected members of the three consanguineous families lies between markers D11S1989 and D11S4176 harbouring the as yet unknown Papillon-Lefèvre syndrome gene. Haplotype analyses in all the families studied support this localisation. This study has identified a further locus harbouring a gene for palmoplantar keratoderma and one possibly involved in periodontitis.

Epigenetic targeting in the mouse zygote marks DNA for later methylation : a mechanism for maternal effects in development

The transgenic sequences in the mouse line TKZ751 are demethylated on a DBA/2 inbred strain background but become highly methylated at postimplantation stages in offspring of a cross with a BALB/c female. In the reciprocal cross the transgene remains demethylated suggesting that imprinted BALB/c methylation modifiers or egg cytoplasmic factors are responsible for this striking maternal effect on de novo methylation. Reciprocal pronuclear transplantation experiments were carried out to distinguish between these mechanisms. The results indicate that a maternally-derived oocyte cytoplasmic factor from BALB/c marks the TKZ751 sequences at fertilization; this mark and postzygotic BALB/c modifiers are both required for de novo methylation of the target sequences at postimplantation stages. Using genetic linkage analyses we mapped the maternal effect to a locus on chromosome 17. Moreover, seven postzygotic modifier loci were identified that increase the postimplantation level of methylation. Analysis of interactions between the maternal and the postzygotic loci shows that both are needed for de novo methylation in the offspring. The combined experiments thus reveal a novel epigenetic marking process at fertilization which targets DNA for later methylation in the foetus. The most significant consequence is that the genotype of the mother can influence the epigenotype of the offspring by this marking process. A number of parental and imprinting effects may be explained by this epigenetic marking.

Cloning of the mouse dysferlin gene and genomic characterization of the SJL-Dysf mutation.

The SJL mouse strain has been widely used as an animal model for experimental autoimmune encephalitis (EAE), inflammatory muscle disease and lymphomas and has also been used as a background strain for the generation of animal models for a variety of diseases including motor neurone disease, multiple sclerosis and atherosclerosis. Recently the SJL mouse was shown to have myopathy due to dysferlin deficiency, so that it can now be considered a natural animal model for limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). We have cloned the mouse dysferlin cDNA and analysis of the sequence shows that the mouse dysferlin gene is characterized by six C2 domain sequences and a C-terminal anchoring domain, with the human and the mouse dysferlin genes sharing > 90% sequence homology overall. Genomic analysis of the SJL mutation confirms that the 171 bp RNA deletion has arisen by exon skipping resulting from a splice site mutation. The identification of this mutation has implications for the various groups using this widely available mouse stock.

Phenotypic differences in Angelman syndrome patients : Imprinting mutations show less frequently microcephaly and hypopigmentation than deletions

Angelman syndrome (AS) is a relatively frequent disorder of psychomotor development caused by loss of function of a gene from chromosome 15q11-q13, a region subject to genomic imprinting. The AS gene(s) is exclusively expressed from the maternal chromosome. Several kinds of mutations have been found to cause AS. More than half of the cases exhibit a deletion of the maternal 15q11-q13 region. Recently, we and others described a new mutation type, the imprinting mutation, characterised by normal, biparental inheritance but aberrant methylation patterns of the entire chromosomal region. In AS, a paternal imprint is found on the maternal chromosome probably leading to functional inactivation of the AS gene(s). We have now compared the phenotype of 9 AS patients with imprinting mutation to that of nine age-matched ones with a maternally derived deletion. Both groups were evaluated for 19 common AS symptoms. All patients, independently of their molecular findings, showed classical AS symptoms such s mental retardation, delayed motor development, and absent speech. In contrast, for two signs, hypopigmentation and microcephaly, a different distribution among both groups was observed. Only one of nine AS patients with an imprinting mutation, but seven of nine in the deletion control group showed either symptom. Our results suggest that imprinting mutations, in contrast to deletions, cause only incomplete loss of gene function or that maternally derived deletions affect also genes not subject to genomic imprinting. We conclude that AS is caused by loss of function of a major gene that is imprinted but that there are also other genes that contribute to the phenotype when in hemizygous condition.