Mutations affecting the BHLHA9 DNA-binding domain cause MSSD, mesoaxial synostotic syndactyly with phalangeal reduction, Malik-Percin type.

Mesoaxial synostotic syndactyly, Malik-Percin type (MSSD) (syndactyly type IX) is a rare autosomal-recessive nonsyndromic digit anomaly with only two affected families reported so far. We previously showed that the trait is genetically distinct from other syndactyly types, and through autozygosity mapping we had identified a locus on chromosome 17p13.3 for this unique limb malformation. Here, we extend the number of independent pedigrees from various geographic regions segregating MSSD to a total of six. We demonstrate that three neighboring missense mutations affecting the highly conserved DNA-binding region of the basic helix-loop-helix A9 transcription factor (BHLHA9) are associated with this phenotype. Recombinant BHLHA9 generated by transient gene expression is shown to be located in the cytoplasm and the cell nucleus. Transcription factors 3, 4, and 12, members of the E protein (class I) family of helix-loop-helix transcription factors, are highlighted in yeast two-hybrid analysis as potential dimerization partners for BHLHA9. In the presence of BHLHA9, the potential of these three proteins to activate expression of an E-box-regulated target gene is reduced considerably. BHLHA9 harboring one of the three substitutions detected in MSSD-affected individuals eliminates entirely the transcription activation by these class I bHLH proteins. We conclude that by dimerizing with other bHLH protein monomers, BHLHA9 could fine tune the expression of regulatory factors governing determination of central limb mesenchyme cells, a function made impossible by altering critical amino acids in the DNA binding domain. These findings identify BHLHA9 as an essential player in the regulatory network governing limb morphogenesis in humans.

Fifth finger camptodactyly maps to chromosome 3q11.2-q13.12 in a large German kindred.

Camptodactyly (MIM 114200) is a digit deformity characterised by permanent flexion contracture of fifth fingers at the proximal interphalangeal (PIP) joints. The sporadic cases are common but a familial occurrence is not much appreciated. In an attempt to identify the genetic basis of camptodactyly, we have analysed a large German family with camptodactyly segregating in an autosomal dominant fashion. The affected family members exhibited clinical features of fifth finger camptodactyly and knuckle pads on the crooked fifth finger and on fingers 2-3. Typically, women were more severely affected than men. Microsatellite analyses of five candidate loci known to be associated with camptodactyly-like phenotypes did not show co-segregation with the phenotype in our family. A genome-wide linkage scan using a total of 414 microsatellite markers gave significant evidence of linkage between the familial phenotype and chromosomal locus 3q11.2-q13.12 (maximum two-point LOD score 3.04). The key recombination events showed that the phenotype localises between markers D3S2465 and D3S3044, spanning an interval of approximately 15 cM. This study reports the first genetic locus linked to isolated autosomal dominant fifth finger camptodactyly with knuckle pads and proves the hypothesis that camptodactyly is distinct from camptodactyly-associated phenotypes including Dupuytren contracture. Additional studies of other families will be necessary to determine the existence of genetic homogeneity or heterogeneity of the anomaly and to narrow down the genetic interval to identify the responsible gene. Since genetic heterogeneity for isolated camptodactyly is likely, we propose to designate the 3q11.2-q13.12 locus as CAMPD1 (ie, camptodactyly 1).

Synpolydactyly and HOXD13 polyalanine repeat: addition of 2 alanine residues is without clinical consequences.

BACKGROUND: Type II syndactyly or synpolydactyly (SPD) is clinically very heterogeneous, and genetically three distinct SPD conditions are known and have been designated as SPD1, SPD2 and SPD3, respectively. SPD1 type is associated with expansion mutations in HOXD13, resulting in an addition of > or = 7 alanine residues to the polyalanine repeat. It has been suggested that expansions < or = 6 alanine residues go without medical attention, as no such expansion has ever been reported with the SPD1 phenotype. METHODS: We describe a large Pakistani and an Indian family with SPD. We perform detailed clinical and molecular analyses to identify the genetic basis of this malformation. RESULTS: We have identified four distinct clinical categories for the SPD1 phenotype observed in the affected subjects in both families. Next, we show that a milder foot phenotype, previously described as a separate entity, is in fact a part of the SPD1 phenotypic spectrum. Then, we demonstrate that the phenotype in both families segregates with an identical expansion mutation of 21 bp in HOXD13. Finally, we show that the HOXD13 polyalanine repeat is polymorphic, and the expansion of 2 alanine residues, evident in unaffected subjects of both families, is without clinical consequences. CONCLUSION: It is the first molecular evidence supporting the hypothesis that expansion of < or = 6 alanine residues in the HOXD13 polyalanine repeat is not associated with the SPD1 phenotype.

Evidence for clinical and genetic heterogeneity of syndactyly type I: the phenotype of second and third toe syndactyly maps to chromosome 3p21.31.

There is good evidence from the medical literature that type I syndactyly, the most common form of the nonsyndromic syndactylies, is clinically heterogeneous. We therefore propose to group the condition into four subtypes, which are all autosomal dominantly inherited. Subtype 1, zygodactyly (cutaneous webbing of second and third toe without hand involvement) is the mildest and most common form. The phenotype varies from unilateral minor impression of webbing to bilateral complete webbing of second and third toe including a fusion of nails. Bony involvement is never observed. Subtype 2 is characterized by bilateral cutaneous and/or bony webbing of third and fourth finger, and second and third toe. The phenotype maps on chromosome 2q34-q36 and was designated as SD1 (ie syndactyly 1). The hallmark of subtype 3 is bilateral cutaneous or bony webbing of third and fourth finger, while subtype 4 shows bilateral cutaneous webbing of fourth and fifth toe. Both, subtype 3 and 4, are rare entities. Here, we present clinical and molecular data of a large Pakistani family with zygodactyly that was mapped to a new locus on chromosome 3p21.31 by genome-wide linkage analysis. The highest LOD score (Zmax=3.38) was obtained with microsatellite marker D3S2409. The disease interval is flanked by markers Chr3_4919 and Chr3_4940 encompassing about 0.20 Mb. Since the same phenotype appears not to be linked to this locus in a German family, we predict genetic heterogeneity in zygodactyly and propose to designate the 3p21.31 locus as ZD1 (i.e., zygodactyly 1).

Autosomal recessive mesoaxial synostotic syndactyly with phalangeal reduction maps to chromosome 17p13.3.

Previously we have described a novel and distinct form of non-syndromic osseous syndactyly segregating in an autosomal recessive pattern in a consanguineous Pakistani family. The limb findings include mesoaxial reduction of the fingers, synostoses of the third and fourth metacarpals with associated single phalanges, fifth finger clinodactyly, and preaxial webbing of toes. We identified another published report of this phenotype in a large, inbred Turkish family. In the present study we mapped the phenotype in the Pakistani and Turkish families to chromosome 17p13.3 (multipoint LOD score 5.1). The identification of a single locus for this complex limb malformation in two families with distinct ethnic backgrounds supports the hypothesis that this is a distinct form of syndactyly. Since this form of syndactyly is phenotypically distinct from the previously described eight types, we propose to name this phenotype mesoaxial synostotic syndactyly with phalangeal reduction (MSSD, type IX syndactyly, Malik-Percin type).

Public, expert and patients' opinions on preimplantation genetic diagnosis (PGD) in Germany.

The regulation of reproductive medicine technologies differs significantly among Western industrialized countries. In Germany, preimplantation genetic diagnosis (PGD) is prohibited due to the Embryo Protection Act, which came into force in 1991. In the last 5 years, this prohibition has been vigorously debated. In the present studies, which are part of the German research programme on ethical implications of the Human Genome Project, representative surveys were undertaken to assess the attitudes on PGD in the general population (n = 1017), five relevant expert groups (n = 879), high genetic risk couples (n = 324) and couples undergoing IVF (n = 108). All groups surveyed clearly favoured allowing PGD in Germany. Compared with the results of recently conducted population surveys in the UK and the USA, where PGD is already carried out, public approval of PGD does not differ significantly. The influence of restrictive biopolitics on the apparently liberal public opinion towards new reproductive technology seems to be marginal according to the present data, which should carefully be considered in the ongoing legislation process on human reproduction.

A novel type of autosomal recessive syndactyly: clinical and molecular studies in a family of Pakistani origin.

Non-syndromic syndactylies have been classified into five major types (I-V), all showing autosomal dominant mode of inheritance. Later, the classification was extended and three additional variants (VI-VIII) were defined. Type VII, the Cenani-Lenz syndactyly, is the only non-syndromic, autosomal recessive type. It is characterized by fusion of all phalanges with metacarpal synostosis, dislocated and dysplastic carpals and infrequently, radio-ulnar fusion. Here, we present a Pakistani family with a novel non-syndromic autosomal recessive syndactyly manifesting a unique combination of clinical features. In both hands, reduction of certain phalanges is evident. Radiological examination shows synostosis of third and fourth metacarpals bearing single phalanges. The first three toes are webbed, with hypoplastic terminal phalanx in all the toes. Besides Cenani-Lenz syndactyly, the phenotype segregating in our family is the second well-documented autosomal recessive, non-syndromic syndactyly. A phenotype similar to our family was described in a Turkish kindred but was considered to be a homozygous expression of type I syndactyly. Since the clinical features in our family had minimal overlap with syndactyly types I, II, and III, we have performed microsatellite marker screening to look for the cosegregation of this phenotype with any of the known loci for these respective types. We show that the phenotype in our family is not linked to chromosomal regions 2q34-q36, 2q31, and 6q22-q23 encompassing loci for syndactyly types I, II, and III.

Muscle pathology in 57 patients with myotonic dystrophy type 2.

We evaluated muscle biopsies from 57 patients with genetically confirmed myotonic dystrophy type 2/proximal myotonic myopathy (DM2/PROMM). Light microscopy showed myopathic together with "denervation-like" changes in almost all biopsies obtained from four different muscles: increased fiber size variation, internal nuclei, small angulated fibers, pyknotic nuclear clumps, and predominant type 2 fiber atrophy. Quantitative morphometry in 18 biopsies that were immunostained for myosin heavy chain confirmed a predominance of nonselective type 2 fiber atrophy. These histological changes were similar in all patients regardless of the site of biopsy, the predominant clinical symptoms and signs, and the clinical course. It is likely that, in a number of undiagnosed patients, DM2 is the underlying disorder. With a better understanding of the histopathological pattern in DM2, biopsies from patients with undiagnosed neuromuscular disorders can now be reevaluated.

Facioscapulohumeral muscular dystrophy. Phenotype-genotype correlation in patients with borderline D4Z4 repeat numbers.

Facioscapulohumeral muscular dystrophy (FSHD) is associated with a decreased number of D4Z4 repeats on chromosome 4q35. Diagnostic difficulties arise from atypical clinical presentations and from an overlap in D4Z4 numbers between controls and FSHD individuals. Thus, a molecular genetic test result with a borderline D4Z4 number has its limitations for the clinician wanting to differentiate between the diagnosis of FSHD and a myopathy presenting with FSHD-like symptoms.To investigate this problem in more detail we conducted a systematic study of 39 unrelated FSHD patients with borderline D4Z4 repeat numbers and 102 healthy controls. Our aim was threefold: [1] to define the molecular diagnostic cut-off point between FSHD cases and the control population, [2] to describe the myopathic spectrum in patients with borderline D4Z4 repeat numbers and [3] to look for correlations between D4Z4 number and clinical severity. The results indicate that there is no definite D4Z4 diagnostic cut-off point separating FSHD, FSHD-like myopathies and controls. A broad myopathic spectrum with four phenotypes (typical FSHD, facial-sparing FSHD, FSHD with atypical features, non-FSHD muscle disease) was found in the borderline region. The expected correlation of D4Z4 repeat number and clinical severity was not found. Therefore the molecular test is of limited help to differentiate FSHD from FSHDlike muscle disorders when the D4Z4 number is n = >or= 8.

Evaluation of BMP4 and its specific inhibitor NOG as candidates in human neural tube defects (NTDs).

Neural tube defects (NTD) are among the most common congenital malformations in humans. The current view is that there are no major genes causing NTDs, but combinations of sequence variants in different genes have additive effects on determining the malformation. Therefore it is important to identify such sequence variants to get a better understanding of NTD pathogenesis. Studies on animal models have shown that BMP4 and NOG are involved in the patterning of the neural tube. We therefore performed a single-strand conformation analysis (SSCA) mutation screen for both genes in 179 spina bifida aperta (SBA) patients. Our SSCA screen revealed four missense mutations in BMP4 and one in NOG. It is likely that these mutations have acted together with other gene variants in independently segregating loci as susceptibility factors in these SBA cases. In addition, a case-control association study provides evidence for a genotype disequilibrium of BMP4 polymorphism 455T-->C (V152A) in exon 5. The frequency of the heterozygous 455TC genotype is lower in cases than in controls (nominal P=0.017), although allele frequencies are similar in both groups. A possible explanation for this finding might be that BMP4 455TC heterozygosity at this site is a protective factor in the normal population, although this hypothesis cannot be proven to date.

A polymorphic microsatellite that mediates induction of PIG3 by p53.

The gene PIG3 is induced by the tumor suppressor p53 but not by p53 mutants unable to induce apoptosis, suggesting its involvement in p53-mediated cell death. Here we show that p53 directly binds and activates the PIG3 promoter, but not through the previously described DNA element. Instead, p53 interacts with a pentanucleotide microsatellite sequence within the PIG3 promoter (TGYCC)n where Y=C or T. Despite its limited similarity to the p53-binding consensus, this sequence is necessary and sufficient for transcriptional activation of the PIG3 promoter by p53 and binds specifically to p53 in vitro and in vivo. In a population of 117 healthy donors from Germany, the microsatellite was found to be polymorphic, the number of pentanucleotide repeats being 10, 15, 16 or 17, and the frequency of alleles 5.1%, 62.0%, 21.4% and 11.5%, respectively. The number of repeats directly correlated with the extent of transcriptional activation by p53. This is the first time that a microsatellite has been shown to mediate the induction of a promoter through direct interaction with a transcription factor. Moreover, this sequence of PIG3 is the first p53-responsive element found to be polymorphic. Inheritance of this microsatellite may affect an individual's susceptibility to cancer.