Congenital Neuronal Ceroid Lipofuscinosis with a Novel CTSD Gene Mutation: A Rare Cause of Neonatal-Onset Neurodegenerative Disorder.

Neuronal ceroid lipofuscinoses represent a heterogeneous group of early onset neurodegenerative disorders that are characterized by progressive cognitive and motor function decline, visual loss, and epilepsy. The age of onset has been historically used for the phenotypic classification of this group of disorders, but their molecular genetic delineation has now enabled a better characterization, demonstrating significant genetic heterogeneity even among individuals with a similar phenotype. The rare Congenital Neuronal Ceroid Lipofuscinosis (CLN10) caused by mutations in the CTSD gene encoding for cathepsin D is associated with a dramatic presentation with onset before or around birth. We report on a female born to consanguineous parents who presented at birth with severe neonatal encephalopathy with massive cerebral and cerebellar shrinking on magnetic resonance imaging. Whole exome sequencing with targeted bioinformatic analysis of a panel of genes associated with prenatal/perinatal onset of neurodegenerative disease was performed and revealed the presence of a novel homozygous in-frame deletion in CTSD. Additional functional studies further confirmed the pathogenic character of this variant and established the diagnosis of CLN10 in the patient.

Experience of a multidisciplinary task force with exome sequencing for Mendelian disorders.

BACKGROUND: In order to optimally integrate the use of high-throughput sequencing (HTS) as a tool in clinical diagnostics of likely monogenic disorders, we have created a multidisciplinary "Genome Clinic Task Force" at the University Hospitals of Geneva, which is composed of clinical and molecular geneticists, bioinformaticians, technicians, bioethicists, and a coordinator. METHODS AND RESULTS: We have implemented whole exome sequencing (WES) with subsequent targeted bioinformatics analysis of gene lists for specific disorders. Clinical cases of heterogeneous Mendelian disorders that could potentially benefit from HTS are presented and discussed during the sessions of the task force. Debate concerning the interpretation of identified variants and the content of the final report constitutes a major part of the task force's work. Furthermore, issues related to bioethics, genetic counseling, quality control, and reimbursement are also addressed. CONCLUSIONS: This multidisciplinary task force has enabled us to create a platform for regular exchanges between all involved experts in order to deal with the multiple complex issues related to HTS in clinical practice and to continuously improve the diagnostic use of HTS. In addition, this task force was instrumental to formally approve the reimbursement of HTS for molecular diagnosis of Mendelian disorders in Switzerland.

Multiplex targeted high-throughput sequencing for Mendelian cardiac disorders.

Mendelian cardiomyopathies and arrhythmias are characterized by an important genetic heterogeneity, rendering Sanger sequencing very laborious and expensive. As a proof of concept, we explored multiplex targeted high-throughput sequencing (HTS) as a fast and cost-efficient diagnostic method for individuals suffering from Mendelian cardiac disorders. We designed a DNA capture assay including all exons from 130 genes involved in cardiovascular Mendelian disorders and analysed simultaneously four samples by multiplexing. Two patients had familial hypertrophic cardiomyopathy (HCM) and two patients suffered from long QT syndrome (LQTS). In patient 1 with HCM, we identified two known pathogenic missense variants in the two most frequently mutated sarcomeric genes MYH7 and MYBPC. In patient 2 with HCM, a known acceptor splice site variant in MYBPC3 was found. In patient 3 with LQTS, two missense variants in the genes SCN5A and KCNQ were identified. Finally, in patient 4 with LQTS a known missense variant was found in MYBPC3, which is usually mutated in patients with cardiomyopathy. Our results showed that multiplex targeted HTS works as an efficient and cost-effective tool for molecular diagnosis of heterogeneous disorders in clinical practice and offers new insights in the pathogenesis of these complex diseases.

Mitotic errors in somatic cells cause trisomy 21 in about 4.5% of cases and are not associated with advanced maternal age.

The study of DNA polymorphisms has permitted the determination of the parental and meiotic origin of the supernumerary chromosome 21 in families with free trisomy 21. Chromosomal segregation errors in somatic cells during mitosis were recognized after analysis of DNA markers in the pericentromeric region and (in order to identify recombination events) along the long arm of chromosome 21. Mitotic errors accounted for about 4.5% (11 of 238) of free trisomy 21 cases examined. The mean maternal age of mitotic errors was 28.5 years and there was no association with advanced maternal age. There was no preference in the parental origin of the duplicated chromosome 21. The 43 maternal meiosis II errors in this study had a mean maternal age of 34.1 years-the highest mean maternal age of all categories of chromosomal segregation errors.

Schizophrenia susceptibility loci on chromosomes 13q32 and 8p21.

Schizophrenia is a common disorder characterized by psychotic symptoms; diagnostic criteria have been established. Family, twin and adoption studies suggest that both genetic and environmental factors influence susceptibility (heritability is approximately 71%; ref. 2), however, little is known about the aetiology of schizophrenia. Clinical and family studies suggest aetiological heterogeneity. Previously, we reported that regions on chromosomes 22, 3 and 8 may be associated with susceptibility to schizophrenia, and collaborations provided some support for regions on chromosomes 8 and 22 (refs 9-13). We present here a genome-wide scan for schizophrenia susceptibility loci (SSL) using 452 microsatellite markers on 54 multiplex pedigrees. Non-parametric linkage (NPL) analysis provided significant evidence for an SSL on chromosome 13q32 (NPL score=4.18; P=0.00002), and suggestive evidence for another SSL on chromosome 8p21-22 (NPL=3.64; P=0.0001). Parametric linkage analysis provided additional support for these SSL. Linkage evidence at chromosome 8 is weaker than that at chromosome 13, so it is more probable that chromosome 8 may be a false positive linkage. Additional putative SSL were noted on chromosomes 14q13 (NPL=2.57; P=0.005), 7q11 (NPL=2.50, P=0.007) and 22q11 (NPL=2.42, P=0.009). Verification of suggestive SSL on chromosomes 13q and 8p was attempted in a follow-up sample of 51 multiplex pedigrees. This analysis confirmed the SSL in 13q14-q33 (NPL=2.36, P=0.007) and supported the SSL in 8p22-p21 (NPL=1.95, P=0.023).

Mutations in DNAH5 account for only 15% of a non-preselected cohort of patients with primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is characterised by recurrent infections of the upper respiratory airways (nose, bronchi, and frontal sinuses) and randomisation of left-right body asymmetry. To date, PCD is mainly described with autosomal recessive inheritance and mutations have been found in five genes: the dynein arm protein subunits DNAI1, DNAH5 and DNAH11, the kinase TXNDC3, and the X-linked retinitis pigmentosa GTPase regulator RPGR. METHODS: We screened 89 unrelated individuals with PCD for mutations in the coding and splice site regions of the gene DNAH5 by denaturing high performance liquid chromatography (DHPLC) and sequencing. Patients were mainly of European origin and were recruited without any phenotypic preselection. RESULTS: We identified 18 novel (nonsense, splicing, small deletion and missense) and six previously described mutations. Interestingly, these DNAH5 mutations were mainly associated with outer + inner dyneins arm ultrastructural defects (50%). CONCLUSION: Overall, mutations on both alleles of DNAH5 were identified in 15% of our clinically heterogeneous cohort of patients. Although genetic alterations remain to be identified in most patients, DNAH5 is to date the main PCD gene.

Epistatic interactions with a common hypomorphic RET allele in syndromic Hirschsprung disease.

Hirschsprung disease (HSCR) stands as a model for genetic dissection of complex diseases. In this model, a major gene, RET, is involved in most if not all cases of isolated (i.e., nonsyndromic) HSCR, in conjunction with other autosomal susceptibility loci under a multiplicative model. HSCR susceptibility alleles can harbor either heterozygous coding sequence mutations or, more frequently, a polymorphism within intron 1, leading to a hypomorphic RET allele. On the other hand, about 30% of HSCR are syndromic. Hitherto, the disease causing gene has been identified for eight Mendelian syndromes with HSCR: congenital central hypoventilation (CCHS), Mowat-Wilson (MWS), Bardet-Biedl (BBS), Shah-Waardenburg (WS4), cartilage-hair-hypoplasia (CHH), Smith-Lemli-Opitz (SLO), Goldberg-Sprintzsen (GSS), and hydrocephalus due to congenital stenosis of the aqueduct of sylvius (HSAS). According to the HSCR syndrome, the penetrance of HSCR trait varies from 5 to 70%. Trisomy 21 (T21) also predisposes to HSCR. We were able to collect a series of 393 patients affected by CCHS (n = 173), WS4 (n = 24), BBS (n = 51), MWS (n = 71), T21 (n = 46), and mental retardation (MR) with HSCR (n = 28). For each syndrome, we studied the RET locus in two subgroups of patients; i.e., with or without HSCR. We genotyped the RET locus in 393 patients among whom 195 had HSCR, and compared the distribution of alleles and genotypes within the two groups for each syndrome. RET acts as a modifier gene for the HSCR phenotype in patients with CCHS, BBS, and Down syndrome, but not in patients with MWS and WS4. The frequent, low penetrant, predisposing allele of the RET gene can be regarded as a risk factor for the HSCR phenotype in CCHS, BBS, and Down syndrome, while its role is not significant in MWS and WS4. These data highlight the pivotal role of the RET gene in both isolated and syndromic HSCR.

[The contribution of molecular genetics to clinical cardiology: the example of hypertrophic cardiomyopathy]. / L'apport de la génétique moléculaire en cardiologie clinique: l'exempte de la cardiomyopathie hypertrophique.

Recent advances in molecular genetics have resulted in the identification of pathogenic mutations in a number of genes which cause hypertrophic cardiomyopathy (HCM). In order to integrate this increasing genetic knowledge of HCM into the cardiology clinic, we offer all patients and their families diagnosis and genetic counselling based on these current data. In addition, within the framework of a multidisciplinary project between the Divisions of Medical Genetics, Cardiology and Pediatric Cardiology of the University Hospitals of Geneva, we have developed a resequencing array enabling rapid molecular diagnosis of HCM. Data from this study will enhance our understanding of the aetiology of HCM, and improve our knowledge of genotype-phenotype correlations. This information will enable us to develop new therapeutic and preventive concepts, with the aim of tailoring therapies to the specific genetic variant of each patient and its family.

Carney complex, Peutz-Jeghers syndrome, Cowden disease, and Bannayan-Zonana syndrome share cutaneous and endocrine manifestations, but not genetic loci.

Carney complex (CC), Peutz-Jeghers syndrome (PJS), Cowden disease (CD), and Bannayan-Zonana syndrome (BZS) share clinical features, such as mucocutaneous lentigines and multiple tumors (thyroid, breast, ovarian, and testicular neoplasms), and autosomal dominant inheritance. A genetic locus has been identified for CC on chromosome 2 (2p16), and the genes for PJS, CD, and BZS were recently identified; genetic heterogeneity appears likely in both CC and PJS. The genes for PJS and CD/BZS, STK11/LKB1 and PTEN, respectively, may act as tumor suppressors, because loss of heterozygosity (LOH) of the PJS and CD/BZS loci has been demonstrated in tumors excised from patients with these disorders. We studied 2 families with CC in whom the disease could not be shown to segregate with polymorphic markers from the 2p16 locus. Their members presented with lesions frequently seen in PJS and the other lentiginosis syndromes. We also tested 16 tumors and cell lines established from patients with CC for LOH involving the PJS and CD/BZS loci. DNA was extracted from peripheral blood, tumor cell lines, and tissues and subjected to PCR amplification with primers from microsatellite sequences flanking the STK11/LKB1 and PTEN genes on 19p13 and 10q23, respectively, and a putative PJS locus on 19q13. All loci were excluded as candidates in both families with LOD scores less than 2 and/or by haplotype analysis. LOH for these loci was not present in any of the tumors that were histologically identical to those seen in PJS. The overall rate of LOH for the PJS and CD/BZS loci in tumors from patients with CC was less than 10%. We conclude that despite substantial clinical overlap among CC, PJS, CD, and BZS, LOH for the STK11 and PTEN loci is an infrequent event in CC-related tumors. Linkage analysis excluded the PJS and CD/BZS loci on chromosomes 19 (19p13 and 19q13) and 10 (10q23) from harboring the gene defect(s) responsible for the phenotype in these 2 families.

An apparently dominant bipolar affective disorder (BPAD) locus on chromosome 20p11.2-q11.2 in a large Turkish pedigree.

Bipolar affective disorder (BPAD), also known as manic-depressive illness, is a common complex, polygenic disorder characterised by recurrent cyclic episodes of mania and depression. Family, twin, and adoption studies strongly suggest a genetic predisposition/susceptibility to BPAD, but no genes have yet been identified. We studied a large Turkish pedigree, with an apparently autosomal dominant BPAD, which contained 13 affected individuals. The age of onset ranged from 15-40 with a mean of 25 years. The phenotypes consisted of recurrent manic and major depressive episodes, including suicidal attempts; there was usually full remission with lithium treatment. A genome-wide linkage analysis using a dominant mode of inheritance showed strong evidence for a BPAD susceptibility locus on chromosome 20p11.2-q11.2. The highest 2-point lod score of 4.34 at theta = 0 was obtained with markers D20S604, D20S470, D20S836 and D20S838 using a dominant model with full penetrance. Haplotype analysis enabled the mapping of the BPAD locus in this family between markers D20S186 and D20S109, to a region of approximately 42 cM.

Molecular mapping of twenty-four features of Down syndrome on chromosome 21.

To determine which regions of chromosome 21 are involved in the pathogenesis of specific features of Down syndrome, we analysed, phenotypically and molecularly, 10 patients with partial trisomy 21. Six minimal regions for 24 features were defined by genotype-phenotype correlations. Nineteen of these features could be assigned to just 2 regions: short stature, joint hyperlaxity, hypotonia, major contribution to mental retardation and 9 anomalies of the face, hand and foot to the region D21S55, or Down syndrome chromosome region (DCR), located on q22.2 or very proximal q22.3, and spanning 0.4-3 Mb; 6 facial and dermatoglyphic anomalies to the region D21S55-MX1, including the DCR and spanning a maximum of 6 Mb on q22.2 and part of q22.3. Thus, the complex phenotype that constitutes Down syndrome may in large part simply result from the overdosage of only one or a few genes within the DCR and/or region D21S55-MX1.

Mapping the Down syndrome chromosome region. Establishment of a YAC contig spanning 1.2 megabases.

The triplication of a region of chromosome 21 around D21S55 in 21q22.2-22.3 has been involved in the main features of Down syndrome including mental retardation (Down syndrome chromosome region: DCR). To improve the physical map of this region, we screened yeast artificial chromosome (YAC) libraries with ETS2 and ERG sequences. Five selected clones were analyzed by AluPCR, pulsed-field gel electrophoresis, and in situ hybridization. A 1.2-Mg contig, encompassing the protooncogenes ETS2 and ERG, was identified, its restriction map established and compared to the genomic map. ERG is distal to D21S55 and proximal to ETS2. ERG and ETS2 genes are 400 kb apart and in opposite orientations. The contig contains the distal boundary and part of the DCR. Three putative HTF islands were identified.

Primary ciliary dyskinesia: a genome-wide linkage analysis reveals extensive locus heterogeneity.

Primary ciliary dyskinesia (PCD), or immotile cilia syndrome (ICS), is an autosomal recessive disorder affecting ciliary movement with an incidence of 1 in 20000-30000. Dysmotility to complete immotility of cilia results in a multisystem disease of variable severity with recurrent respiratory tract infections leading to bronchiectasis and male subfertility. Ultrastructural defects are present in ciliated mucosa and spermatozoa. Situs inversus (SI) is found in about half of the patients (Kartagener syndrome). We have collected samples from 61 European and North American families with PCD. A genome-wide linkage search was performed in 31 multiplex families (169 individuals including 70 affecteds) using 188 evenly spaced (19cM average interval) polymorphic markers. Both parametric (recessive model) and non-parametric (identity by descent allele sharing) linkage analyses were used. No major locus for the majority of the families was identified, although the sample was powerful enough to detect linkage if 40% of the families were linked to one locus. These results strongly suggest extensive locus heterogeneity. Potential genomic regions harbouring PCD loci were localised on chromosomes 3p, 4q, 5p, 7p, 8q, 10p, 11q, 13q, 15q, 16p, 17q and 19q. Linkage analysis using PCD families with a dynein arm deficiency provided 'suggestive' evidence for linkage to chromosomal regions 8q, 16pter, while analyses using only PCD families with situs inversus resulted in 'suggestive' scores for chromosomes 8q, and 19q.

Biparental inheritance of chromosome 21 polymorphic markers indicates that some Robertsonian translocations t(21;21) occur postzygotically.

Robertsonian translocations between acrocentric chromosomes are the most common structural chromosomal rearrangements in humans and many other organisms, and several mechanisms for their formation have been proposed. We have analyzed highly informative DNA polymorphisms in a family with a non-mosaic de novo Robertsonian translocation 21q;21q, to determine the parental origin of the two 21q arms of the rearranged chromosome. The genotypes indicated a biparental origin, i.e. one 21q was paternal and the other maternal. These results imply that in some cases the formation of the rob(21q;21q) occurs in the zygote or in the first few postzygotic mitotic divisions.

An autosomal dominant triphalangeal thumb: polysyndactyly syndrome with variable expression in a large Indian family maps to 7q36.

Hereditary developmental abnormalities of the upper or lower limbs in humans are easily recognizable phenotypes that can be used in the mapping and cloning of genes involved in normal human development. We studied a large Indian pedigree (UR002) with an autosomal dominant triphalangeal thumb (TPT) and polysyndactyly (PSD). The abnormalities were present only in the upper limbs, and the phenotype was fully penetrant. The expression of the phenotype was variable and ranged from unilateral TPT to bilateral TPT, preaxial du-, tri-, or quadruplication of the thumb, or syndactyly of multiple thumbs. There were 112 affected individuals in the pedigree. Previous linkage analyses on apparently similar phenotypes have identified a locus at 7q36 [Heutink et al., 1994, Nature Genet 6:287-291; Tsukurov et al., 1994]. To map the gene responsible for the TPT-PSD in family UR002, we performed linkage analysis in DNA from 47 affected and 7 normal individuals. Marker D7S550, located at 7q36, yielded a maximum LOD score of 11.31 at theta = 0.00. Additional markers in the region also showed no recombination. These data indicate that the gene responsible for the hand abnormality in pedigree UR002 maps to the same region as that in previous pedigrees with similar phenotype. Further analyses of recombinants among all the linked families by using new polymorphic markers will narrow the critical genomic region and facilitate positional cloning of the elusive gene.

Trisomy 12 mosaicism confirmed in multiple organs from a liveborn child.

This patient, in whom trisomy 12 mosaicism was confirmed in multiple organs, is the fifth case diagnosed postnatally and the first reported for whom a meiotic origin of the trisomy, maternal meiosis I, was determined. Mosaic aneuploidy was suspected because of pigmentary dysplasia, a frequent but non-specific finding in chromosomal mosaicism. The severe phenotype of this child, who died in infancy with a complex heart malformation, was probably a result of the high percentage of trisomic cells. Cytogenetic and interphase fluorescent in situ hybridization analyses showed a highly variable distribution of aneuploid cells in the nine tissues studied, from none in blood and ovary to 100% in spleen and liver. The trisomy arose meiotically with apparent post-zygotic loss of one of the chromosomes 12; uniparental disomy for this chromosome in the diploid cell line was excluded. The phenotype of the cases reported in living or liveborn individuals has been extremely variable, ranging from the present case, in which the child died in infancy with multiple malformations and pigmentary dysplasia, to a fortuitous finding in an adult studied for infertility. The variation in severity is probably determined by the proportion and distribution of the trisomic cells, which is linked to the timing of the non-disjunctional error.

Schizophrenia: a genome scan targets chromosomes 3p and 8p as potential sites of susceptibility genes.

Using a systematically ascertained sample of 57 families, each having 2 or more members with a consensus diagnosis of schizophrenia (DSM-III-R criteria), we have carried out linkage studies of 520 loci, covering approximately 70% of the genome for susceptibility loci for schizophrenia. A two-stage strategy based on lod score thresholds from simulation studies of our sample identified regions for further exploration. In each region, a dense map of highly informative dinucleotide repeat polymorphisms (heterozygosity greater than .70) was analyzed using dominant, recessive, and "affected only" models and nonparametric sib pair identity-by-descent methods. For one region, 8p22-p21, affected sib-pair analyses gave a P value = .0001, corresponding to a lod score approximately equal to 3.00. For 8p22-p21, the maximum two-point lod score occurred using the "affected only" recessive model (ZMAX = 2.35; theta M = theta F); allowing for a constant sex difference in recombination fractions found in reference pedigrees, ZMAX = 2.78 (theta M/theta F = 3). For a second region, 3p26-p24, the maximum two-point lod score was 2.34 ("affected only" dominant model), and the affected sib-pair P value was .01. These two regions are worthy of further exploration as potential sites of susceptibility genes for schizophrenia.