<Emphasis Type="Italic">Sonic hedgehog</Emphasis> mutations identified in holoprosencephaly patients can act in a dominant negative manner

Sonic hedgehog (SHH) plays an important instructional role in vertebrate development, as exemplified by the numerous developmental disorders that occur when the SHH pathway is disrupted. Mutations in the SHH gene are the most common cause of sporadic and inherited holoprosencephaly (HPE), a developmental disorder that is characterized by defective prosencephalon development. SHH HPE mutations provide a unique opportunity to better understand SHH biogenesis and signaling, and to decipher its role in the development of HPE. Here, we analyzed a panel of SHH HPE missense mutations that encode changes in the amino-terminal active domain of SHH. Our results show that SHH HPE mutations affect SHH biogenesis and signaling at multiple steps, which broadly results in low levels of protein expression, defective processing of SHH into its active form and protein with reduced activity. Additionally, we found that some inactive SHH proteins were able to modulate the activity of wt SHH in a dominant negative manner, both in vitro and in vivo. These findings show for the first time the susceptibility of SHH driven developmental processes to perturbations by low-activity forms of SHH. In conclusion, we demonstrate that SHH mutations found in HPE patients affect distinct steps of SHH biogenesis to attenuate SHH activity to different levels, and suggest that these variable levels of SHH activity might contribute to some of the phenotypic variation found in HPE patients. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. S. Singh, R. Tokhunts and V. Baubet contributed equally to this work.     

Refined mapping of the gene for autosomal dominant retinitis pigmentosa (RP17) on chromosome 17q22

Linkage analysis was performed on a large Dutch family with autosomal dominant retinitis pigmentosa. Linkage was found to the RP17 locus on chromosome 17q22, which was previously described in two South African families by Bardien et al. (1995, 1997). Assuming that the disease phenotypes in these families are caused by the same gene, the RP17 critical region is refined to a 7.7-cM interval between markers D17S1607 and D17S948. Two positional candidate genes, the retina-specific amine oxidase (RAO) gene (AOC2) and the cone transducin γ gene (GNGT2), were excluded. Received: 7 September 1998 / Accepted: 23 November 1998     

Correlation between Waardenburg syndrome phenotype and genotype in a population of individuals with identified PAX3 mutations

Waardenburg syndrome (WS) type 1 is an autosomal dominant disorder characterized by sensorineural hearing loss, pigmentary abnormalities of the eye, hair, and skin, and dystopia canthorum. The phenotype is variable and affected individuals may exhibit only one or a combination of several of the associated features. To assess the relationship between phenotype and gene defect, clinical and genotype data on 48 families (271 WS individuals) collected by members of the Waardenburg Consortium were pooled. Forty-two unique mutations in the PAX3 gene, previously identified in these families, were grouped in five mutation categories: amino acid (AA) substitution in the paired domain, AA substitution in the homeodomain, deletion of the Ser-Thr-Pro-rich region, deletion of the homeodomain and the Ser-Thr-Pro-rich region, and deletion of the entire gene. These mutation classes are based on the structure of the PAX3 gene and were chosen to group mutations predicted to have similar defects in the gene product. Association between mutation class and the presence of hearing loss, eye pigment abnormality, skin hypopigmentation, or white forelock was evaluated using generalized estimating equations, which allowed for incorporation of a correlation structure that accounts for potential similarity among members of the same family. Odds for the presence of eye pigment abnormality, white forelock, and skin hypopigmentation were 2, 8, and 5 times greater, respectively, for individuals with deletions of the homeodomain and the Pro-Ser-Thr-rich region compared to individuals with an AA substitution in the homeodomain. Odds ratios that differ significantly from 1.0 for these traits may indicate that the gene products resulting from different classes of mutations act differently in the expression of WS. Although a suggestive association was detected for hearing loss with an odds ratio of 2.6 for AA substitution in the paired domain compared with AA substitution in the homeodomain, this odds ratio did not differ significantly from 1.0. Received: 27 July 1997 / Accepted: 9 December 1997     

Hereditary hemorrhagic telangiectasia: ENG and ALK-1 mutations in Dutch patients

Hereditary hemorrhagic telangiectasia (HHT) or Rendu-Osler-Weber disease is an autosomal dominant disorder characterized by an aberrant vascular development. The resulting vascular lesions range from smaller mucocutaneous telangiectases to large visceral arteriovenous malformations, especially in the skin, lung, gastrointestinal tract and the brain. Mutations in the genes encoding endoglin (ENG, chromosome 9q34) and activin A receptor type-like kinase 1 (ALK-1, also named ACVRL1, chromosome 12q13) are associated with HHT1 and HHT2, respectively. We report here on the genetic and molecular heterogeneity found in the HHT population in the Netherlands. Probands of 104 apparently unrelated families were studied and we performed sequence analysis on both the ENG gene and ALK-1 gene. In most of the probands, we found a mutation in one of the two genes: 53% in the ENG gene and 40% in the ALK-1 gene. In 7% of the families no ENG or ALK1 mutation was found. The mutations detected were deletions, insertions, nonsense, missense and splice site mutations. The majority were novel mutations.     

Novel and recurrent tyrosine aminotransferase gene mutations in tyrosinemia typeII

Tyrosinemia typeII (Richner-Hanhart syndrome, RHS) is a disorder of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT). We have previously described one deletion and six different point mutations in four RHS patients. We have now analyzed the TAT genes in a further seven unrelated RHS families from Italy, France, the United Kingdom, and the United States. We have established PCR conditions for the amplification of all twelve TAT exons and have screened the products for mutations by direct sequence analysis or by first performing single-strand conformation polymorphism analysis. We have thus identified the presumably pathological mutations in eight RHS alleles, including two nonsense mutations (R57X, E411X) and four amino acid substitutions (R119W, L201R, R433Q, R433W). Only the R57X mutation, which was found in one Scottish and two Italian families, has been previously reported in another Italian family. Haplotype analysis indicates that this mutation, which involves a CpG dinucleotide hot spot, has a common origin in the three Italian families but arose independently in the Scottish family. Two polymorphisms have also been detected, viz., a protein polymorphism, P15S, and a silent substitution S103S (TCG→TCA). Expression of R433Q and R433W demonstrate reduced activity of the mutant proteins. In all, twelve different TAT gene mutations have now been identified in tyrosinemia typeII. Received: 8 October 1997 / Accepted: 29 October 1997     

<Emphasis Type="Italic">CHX10</Emphasis> mutations cause non-syndromic microphthalmia/anophthalmia in Arab and Jewish kindreds

Microphthalmia/anophthalmia is a clinically heterogeneous disorder of eye formation, ranging from small size of a single eye to complete bilateral absence of ocular tissues. The genetic defect underlying isolated autosomal recessive microphthalmia/anophthalmia is yet unclear. We studied four families (two of Arab origin, one of Bedouin origin, and one of Persian-Jewish origin) with autosomal recessive microphthalmia/anophthalmia and no associated eye anomalies, and one Syrian–Jewish family with associated colobomas. Assuming a founder effect in each of the families, we performed homozygosity mapping using polymorphic markers adjacent to human homologues of genes known to be associated with eye absence in various species, namely EYA1, EYA2, EYA3, SIX4, SIX6, PAX6 and CHX10. No association was found with EYA1, EYA2, EYA3, SIX6 or PAX6. In two families, linkage analysis was consistent with possible association with SIX4, but no mutations were found in the coding region of the gene or its flanking intron sequences. In three of the five families, linkage analysis followed by sequencing demonstrated that affected individuals in each family were homozygous for a different CHX10 aberration: a mutation in the CVC domain and a deletion of the homeobox domain were found in two Arab families, and a mutation in the donor-acceptor site in the first intron in the Syrian-Jewish family. There was phenotypic variation between families having different mutations, but no significant phenotypic variation within each family. It has been previously shown that mutations in a particular nucleotide in CHX10 are associated with an autosomal recessive syndrome of microphthalmia/anophthalmia with iris colobomas and cataracts in two families. We now show that different mutations in other domains of the same gene underlie isolated microphthalmia/anophthalmia.     

New p57KIP2 mutations in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is characterized by numerous growth abnormalities and an increased risk of childhood tumors. The gene for BWS is localized in the 11p15.5 region, as determined by linkage analysis of autosomal dominant pedigrees. The increased maternal transmission pattern seen in the autosomal dominant-type pedigrees and the findings of paternal uniparental disomy reported for a subgroup of patients indicate that the gene for BWS is imprinted. Previously, we found p57 ^KIP2 , which is a Cdk-kinase inhibitor located at 11p15, is mutated in two BWS patients. Here, we screened for the mutation of the gene in 15 BWS patients. Received: 25 March 1997 / Accepted: 22 May 1997     

Phenotypic variability in two families with novel splice-site and frameshift NF2 mutations

Neurofibromatosis 2 (NF2) is a clinically variable autosomal dominant disorder, caused by mutations in the NF2 tumor suppressor gene on chromosome 22q12, that predisposes to nervous system tumors and ocular abnormalities. To assess intrafamilial phenotypic variability, we performed mutation analysis and clinical assessment on two multigeneration NF2 families with five patients and seven asymptomatic first-degree relatives of patients. One family had a point mutation of agCC→ggCC at position 1447–2 at the exon 13/14 boundary predicted to lead to an altered splice acceptor sequence and exon deletion. The other family had an insertion of 2 base pairs (TC) at position 761 in exon 8, leading to a frameshift. Both mild and severe phenotypes occurred in each family, indicating that phenotypic variability in NF2 can be caused by factors other than NF2 mutations. Genetic counseling of NF2 families should include the possibility that presymptomatic NF2 mutation carriers can develop a different phenotype than previously diagnosed patients. Received: 4 January 1996 / Revised: 26 March 1996     

A genetic screen for elements of the network that regulates neurogenesis in Drosophila

The development of external sensory organs on the notum of Drosophila is promoted by the proneural genes achaete and scute. Their activity defines proneural cell clusters in the wing imaginal disc. Ectopic expression, under control of the GAL4 system, of the proneural gene lethal of scute (l'sc) causes the development of ectopic bristles. Persistent ectopic expression of l'sc is not sufficient to impose a neural fate on any given cell. This implies that mutual inhibition, mediated by the Notch signaling pathway, occurs among the cells of the ectopic proneural cluster. Consequently, the dominant, quantifiable phenotype associated with ectopic expression of l'sc is modified by mutations in genes known to be involved in neurogenesis. This phenotype has been utilized to screen for dominant enhancers and suppressors that modify the number of ectopic bristles. In this way, about 100 000 progeny of EMS or X-ray-treated flies have been analyzed to identify autosomal genes involved in regulation of the neural fate. In addition 1200 chromosomes carrying lethal P-element insertions were screened for modifiers. Besides mutations in genes expected to modify the phenotype, we have isolated mutations in six genes not known so far to be involved in neurogenesis. Received: 20 September 1997 / Accepted: 8 October 1997     

Primary Coenzyme Q deficiency Due to Novel ADCK3 Variants,Studies in Fibroblasts and Review of Literature

Primary deficiency of coenzyme Q10 (CoQ10 ubiquinone), is classified as a mitochondrial respiratory chain disorder with phenotypic variability. The clinical manifestation may involve one or multiple tissue with variable severity and presentation may range from infancy to late onset. ADCK3 gene mutations are responsible for the most frequent form of hereditary CoQ10 deficiency (Q10 deficiency-4 OMIM #612016) which is mainly associated with autosomal recessive spinocerebellar ataxia (ARCA2, SCAR9). Here we provide the clinical, biochemical and genetic investigation for unrelated three nuclear families presenting an autosomal form of Spino-Cerebellar Ataxia due to novel mutations in the ADCK3 gene. Using next generation sequence technology we identified a homozygous Gln343Ter mutation in one family with severe, early onset of the disease and compound heterozygous mutations of Gln343Ter and Ser608Phe in two other families with variable manifestations. Biochemical investigation in fibroblasts showed decreased activity of the CoQ dependent mitochondrial respiratory chain enzyme succinate cytochrome c reductase (complex II?+?III). Exogenous CoQ slightly improved enzymatic activity, ATP production and decreased oxygen free radicals in some of the patient’s cells. Our results are presented in comparison to previously reported mutations and expanding the clinical, molecular and biochemical spectrum of ADCK3 related CoQ10 deficiencies.

     

Two novel mutations in exons 19a and 20 and a BsaI polymorphism in a newly characterized intron of the neurofibromatosis type 1 gene

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. It is caused by mutations in the NF1 gene, which comprises 60 exons and is located on chromosome 17q11.2. A total of 170 unrelated NF1 patients were screened for mutations in four exons by temperature-gradient gel electrophoresis. Preparatory work revealed the presence of a previously uncharacterized intron (19a) in what was previously designated exon 19; this allowed us to develop assays for genomic mutation screening in the newly defined exons 19a and 19b. Two novel NF1 mutations were detected: a single-base insertion in exon 19a creating a frameshift, and a second mutation affecting the splice donor site of intron 20 and leading to skipping of exon 20. A novel BsaBI polymorphism was identified in intron 19a. Received: 11 August 1997 / Accepted: 13 November 1997     

Clinical and molecular analysis of the enamelin gene ENAM in Colombian families with autosomal dominant amelogenesis imperfecta

In this study, we analyzed the phenotype, clinical characteristics and presence of mutations in the enamelin gene ENAM in five Colombian families with autosomal dominant amelogenesis imperfecta (ADAI). 22 individuals (15 affected and seven unaffected) belonging to five Colombian families with ADAI and eight individuals (three affected and five unaffected) belonging to three Colombian families with autosomal recessive amelogenesis imperfecta (ARAI) that served as controls for molecular alterations and inheritance patterns were studied. Clinical, radiographic and genetic evaluations were done in all individuals. Eight exons and three intron-exon boundaries were sequenced for mutation analysis. Two of the five families with ADAI had the hypoplasic phenotype, two had the hypocalcified phenotype and one had the hypomaturative phenotype. Anterior open bite and mandibular retrognathism were the most frequent skeletal abnormalities in the families with ADAI. No mutations were found. These findings suggest that ADAI in these Colombian families was unrelated to previously described mutations in the ENAM gene. These results also indicate that other regions not included in this investigation, such as the promoter region, introns and other genes should be considered as potential ADAI candidates.     

Rapid screening of myelin genes in CMT1 patients by SSCP analysis: identification of new mutations and polymorphisms in the P0 gene

Charcot-Marie-Tooth type (CMT1) disease or hereditary motor and sensory neuropathy type I (HMSNI) is an autosomal dominant peripheral neuropathy. In most CMT1 families, the disease cosegregates with a 1.5-Mb duplication on chromosome 17p11.2 (CMT1A). A few patients have been found with mutations in the peripheral myelin protein 22 (PMP-22) gene located in the CMT1A region. In other families mutations have been identified in the major peripheral myelin protein p_o gene localized on chromosome Iq21-q23 (CMT1B). We performed a rapid mutation screening of the PMP-22 and P₀ genes in non-duplicated CMT1 patients by single-strand conformation polymorphism analysis followed by direct polymerase chain reaction sequencing of genomic DNA. Six new single base changes in the P₀ gene were observed: two missense mutations in, respectively, exons 2 and 3, two nonsense mutations in exon 4, and two silent mutations or polymorphisms in, respectively, exons 3 and 6.     

Mapping the multiple self-healing squamous epithelioma (MSSE) gene and investigation of xeroderma pigmentosum group A (XPA) and PATCHED (PTCH) as candidate genes

The MSSE gene predisposes to the development of multiple invasive but self-healing skin tumours (multiple self-healing squamous epitheliomata, MSSE). MSSE (previously named ESS1) was mapped to chromosome 9q by linkage analysis; haplotype analysis in families then suggested a common founder mutation and indicated that the gene lies in the interval D9S1–D9S29 (9q22–q31). Squamous cell carcinomata also develop as one of the complications of xeroderma pigmentosum, and one of the xeroderma pigmentosum genes (XPA) maps within the MSSE interval. We have investigated the hypothesis that a novel dominant mutation in XPA is responsible for MSSE. We screened the entire coding region, 3′ untranslated region (UTR) and 5′UTR of XPA for germline mutations in MSSE families by single-stranded conformation polymorphism analysis and by direct DNA sequencing. No mutations were detected but a novel intragenic polymorphism was identified in the 5′UTR of XPA, in both MSSE-affected and unrelated normal individuals. This XPA polymorphism and nine new polymorphic markers that map in the MSSE region were typed in eleven MSSE families; XPA was excluded as the MSSE gene and the most likely location of MSSE was reduced to the interval between D9S197 and (D9S287, D9S1809). The Patched (PTCH) gene, which is mutated in naevoid basal cell carcinoma syndrome (NBCCS or Gorlin syndrome) lies in this interval and all MSSE families have been shown to share a common haplotype at three novel intragenic PTCH polymorphisms. Although no mutation has been detected in MSSE families, PTCH has not been excluded as the MSSE gene. Received: 6 May 1997 / Accepted: 3 September 1997     

Mutation analysis of hereditary multiple exostoses in the Chinese

Hereditary multiple exostoses (EXT; MIM 133700) is an autosomal dominant bone disorder. It is genetically heterogeneous with at least three chromosomal loci: EXT1 on 8q24.1, EXT2 on 11p11, and EXT3 on 19p. EXT1 and EXT2, the two genes responsible for EXT1 and EXT2, respectively, have been cloned. Recently, three other members of the EXT gene family, named the EXT-like genes (EXTL: EXTL1, EXTL2, and EXTL3), have been isolated. EXT1, EXT2, and the three EXTLs are homologous with one another. We have identified the intron-exon boundaries of EXTL1 and EXTL3 and analyzed EXT1, EXT2, EXTL1, and EXTL3, in 36 Chinese families with EXT, to identify underlying disease-related mutations in the Chinese population. Of the 36 families, five and 12 family groups have mutations in EXT1 and EXT2, respectively. No disease-related mutation has been found in either EXTL1 or EXTL2, although one polymorphism has been detected in EXTL1. Of the 15 different mutations (three families share a common mutation in EXT2), 12 are novel. Most of the mutations are either frameshift or nonsense mutations (12/15). These mutations lead directly or indirectly to premature stop codons, and the mutations generate truncated proteins. This finding is consistent with the hypothesis that the development of EXT is mainly attributable to loss of gene function. Missense mutations are rare in our families, but these mutations may reflect some functionally crucial regions of these proteins. EXT1 is the most frequent single cause of EXT in the Caucasian population in Europe and North America. It accounts for about 40% of cases of EXT. Our study of 36 EXT Chinese families has found that EXT1 seems much less common in the Chinese population, although the frequency of the EXT2 mutation is similar in the Caucasian and Chinese populations. Our findings suggest a possibly different genetic spectrum of this disease in different populations. Electronic Publication     

Gly118Asp is a SCA14 founder mutation in the Dutch ataxia population

Missense mutations in the PRKCG gene have recently been identified in spinocerebellar ataxia 14 (SCA14) patients; these include the Gly118Asp mutation that we found in a large Dutch autosomal dominant cerebellar ataxia (ADCA) family. We subsequently screened the current Dutch ataxia cohort (approximately 900 individuals) for SCA14 mutations in the Cys2 region of the PRKCG gene. We identified the Gly118Asp mutation in another eight individuals from five small families. Haplotype analysis identified a shared chromosomal region surrounding the SCA14 gene, and genealogical research was able to link all these ADCA patients to a single common ancestor. We therefore confirmed that the Gly118Asp mutation is a SCA14 founder mutation in the Dutch ADCA population.     

Use of three DNA polymorphisms of the LDL receptor gene in the diagnosis of familial hypercholesterolemia

Summary Familial hypercholesterolemia (FH) is an autosomal dominant metabolic disorder caused by several different mutations in the low density lipoprotein (LDL) receptor gene. This large number of different mutations, often undetectable in Southern blotting, makes it impossible directly to diagnose the disease. However, restriction fragment length polymorphisms (RFLPs) can be used to follow the inheritance of the defective gene in FH families. In the present study, we report the use of three RFLPs, detected by PvuII, ApaLI and AvaII restriction enzymes, to determine the haplotypes of normal and defective LDL receptor genes in 61 families with FH and in 128 normal individuals. Two of the nine haplotypes were significantly more often associated with the affected genes, whereas one was significantly less frequent. Although none of the associations was strong enough to allow diagnosis in individuals, it was possible, using the three RFLPs, to identify the haplotype of the affected gene in 57 families and to carry out unequivocal diagnosis in 67% of the cases. In four families, PvuII and AvaII detected an abnormal fragment co-segregating with the disease, thus increasing the percentage of diagnosis to 73.4% of the cases.