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Dent disease is a rare X-linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and variable manifestations of other proximal tubule dysfunctions. It often progresses over a few decades to chronic renal insufficiency, and therefore molecular characterization is important to allow appropriate genetic counseling. Two genetic subtypes have been described to date: Dent disease 1 is caused by mutations of the CLCN5 gene, coding for the chloride/proton exchanger ClC-5; and Dent disease 2 by mutations of the OCRL gene, coding for the inositol polyphosphate 5-phosphatase OCRL-1. Herein, we review previously reported mutations (n = 192) and their associated phenotype in 377 male patients with Dent disease 1 and describe phenotype and novel (n = 42) and recurrent mutations (n = 24) in a large cohort of 117 Dent disease 1 patients belonging to 90 families. The novel missense and in-frame mutations described were mapped onto a three-dimensional homology model of the ClC-5 protein. This analysis suggests that these mutations affect the dimerization process, helix stability, or transport. The phenotype of our cohort patients supports and extends the phenotype that has been reported in smaller studies., (© 2015 WILEY PERIODICALS, INC.)

Haemorrhagic stroke accounts for ∼20% of stroke cases and porencephaly is a clinical consequence of perinatal cerebral haemorrhaging. Here, we report the identification of a novel dominant G702D mutation in the collagen domain of COL4A2 (collagen IV alpha chain 2) in a family displaying porencephaly with reduced penetrance. COL4A2 is the obligatory protein partner of COL4A1 but in contrast to most COL4A1 mutations, the COL4A2 mutation does not lead to eye or kidney disease. Analysis of dermal biopsies from a patient and his unaffected father, who also carries the mutation, revealed that both display basement membrane (BM) defects. Intriguingly, defective collagen IV incorporation into the dermal BM was observed in the patient only and was associated with endoplasmic reticulum (ER) retention of COL4A2 in primary dermal fibroblasts. This intracellular accumulation led to ER stress, unfolded protein response activation, reduced cell proliferation and increased apoptosis. Interestingly, the absence of ER retention of COL4A2 and ER stress in cells from the unaffected father indicate that accumulation and/or clearance of mutant COL4A2 from the ER may be a critical modifier for disease development. Our analysis also revealed that mutant collagen IV is degraded via the proteasome. Importantly, treatment of patient cells with a chemical chaperone decreased intracellular COL4A2 levels, ER stress and apoptosis, demonstrating that reducing intracellular collagen accumulation can ameliorate the cellular phenotype of COL4A2 mutations. Importantly, these data highlight that manipulation of chaperone levels, intracellular collagen accumulation and ER stress are potential therapeutic options for collagen IV diseases including haemorrhagic stroke.

Purpose: Nonsyndromic autosomal recessive optic atrophy (arOA) is extremely rare and its existence was disputed until a locus, optic atrophy 6 (OPA6), was mapped to 8q. Recently, a second locus, OPA7, was found on 11q in several families from North Africa, with one presumably ancestral mutation of transmembrane protein 126A (TMEM126A). Here we report an independently ascertained large consanguineous family of Moroccan descent with three siblings affected with nonsyndromic arOA., Methods: Assuming autosomal recessive inheritance, we identified a locus on 11q with homozygosity mapping, with a multipoint logarithm of the odds score of 3.84, and sequenced two candidate genes. Direct sequencing of the complete coding sequence of TMEM126A revealed mutation p.Arg55X, homozygous in all affected siblings and heterozygous in both unaffected parents., Results: This mutation was identical to that recently reported in families from North Africa, consistent with a single ancestral origin. In contrast to the recently reported patients, however, the siblings reported in this study had a relatively mild clinical course, with sudden onset in adolescence in the proband. Interestingly, the proband, but not the other affected siblings, had sensory-motor axonal neuropathy with electrophysiological data strongly suggestive of focal demyelinating abnormalities. An unaffected sibling had transient loss of vision after exercise, i.e., Uhthoff's sign of optic neuropathy, and was found to be a heterozygous carrier of the mutation., Conclusions: Our results confirm genetic heterogeneity in arOA, illustrate clinical variability between families with the p.Arg55X mutation including the description of a mild phenotype in a heterozygote, and underscore the implication of mitochondrial proteins in optic and peripheral neuropathy.

Leber Congenital Amaurosis (LCA), the most severe inherited retinal dystrophy, is genetically heterogeneous, with 14 genes accounting for 70% of patients. Here, 91 LCA probands underwent LCA chip analysis and subsequent sequencing of 6 genes (CEP290, CRB1, RPE65, GUCY2D, AIPL1and CRX), revealing mutations in 69% of the cohort, with major involvement of CEP290 (30%). In addition, 11 patients with early-onset retinal dystrophy (EORD) and 13 patients with Senior-Loken syndrome (SLS), LCA-Joubert syndrome (LCA-JS) or cerebello-oculo-renal syndrome (CORS) were included. Exhaustive re-inspection of the overall phenotypes in our LCA cohort revealed novel insights mainly regarding the CEP290-related phenotype. The AHI1 gene was screened as a candidate modifier gene in three patients with the same CEP290 genotype but different neurological involvement. Interestingly, a heterozygous novel AHI1 mutation, p.Asn811Lys, was found in the most severely affected patient. Moreover, AHI1 screening in five other patients with CEP290-related disease and neurological involvement revealed a second novel missense variant, p.His758Pro, in one LCA patient with mild mental retardation and autism. These two AHI1 mutations might thus represent neurological modifiers of CEP290-related disease., (© 2010 Wiley-Liss, Inc.)

Objectives: To investigate the genetic causes of idiopathic sporadic prenatal generalized edema., Study Design: In a series of 12 patients, in whom in utero generalized skin edema or hydrops fetalis had been diagnosed, we screened 3 lymphangiogenic genes, VEGFR3, FOXC2, and SOX18., Results: In 3 of the patients, we identified a mutation: 2 in VEGFR3 and 1 in FOXC2. Two of the mutations were de novo and one was either de novo or nonpenetrant inherited. In these patients, the generalized edema resorbed spontaneously, either in utero or after birth. In the 2 individuals with a VEGFR3 mutation, edema remained limited to lower limbs., Conclusions: Mutations in the VEGFR3 and FOXC2 genes account for a subset of patients with unexplained in utero generalized subcutaneous edema and hydrops fetalis without family history of lymphedema. Lymphangiogenic genes should be screened for mutations in sporadic patients diagnosed with fetal edema.

Blepharophimosis syndrome (BPES) is caused by loss-of-function mutations in the single-exon forkhead transcription factor gene FOXL2 and by genomic rearrangements of the FOXL2 locus. Here, we focus on 92 new intragenic FOXL2 mutations, 34 of which are novel. Specifically, we found 10 nonsense mutations (11%), 13 missense mutations (14%), 40 deletions or insertions leading to a frameshift (43%), and 29 in-frame changes (32%), of which 28 (30%) lead to a polyalanine expansion. This study confirms the existence of two previously described mutational hotspots. Moreover, we gained novel insights in genotype-phenotype correlations, emphasizing the need to interpret genotype-phenotype correlations individually and always in the context of further clinical observations., ((c) 2008 Wiley-Liss, Inc.)

Harboyan syndrome, or corneal dystrophy and perceptive deafness (CDPD), consists of congenital corneal endothelial dystrophy and progressive perceptive deafness, and is transmitted as an autosomal recessive trait. CDPD and autosomal recessive, non-syndromic congenital hereditary endothelial corneal dystrophy (CHED2) both map at overlapping loci at 20p13, and mutations of SLC4A11 were reported recently in CHED2. A genotype study on six families with CDPD and on one family with either CHED or CDPD, from various ethnic backgrounds (in the seventh family, hearing loss could not be assessed because of the proband's young age), is reported here. Novel SLC4A11 mutations were found in all patients. Why some mutations cause hearing loss in addition to corneal dystrophy is presently unclear. These findings extend the implication of the SLC4A11 borate transporter beyond corneal dystrophy to perceptive deafness.

Objective: To compare the diagnostic accuracy of two-dimensional (2D) ultrasound and three-dimensional (3D) computed tomography (CT) for the diagnosis of fetal skeletal anomalies., Methods: Eleven pregnant women underwent 2D ultrasound and 3D-CT. Ten fetuses presented skeletal anomalies on 2D ultrasound and one fetus had a normal ultrasound exam but a familial history of osteopetrosis. We compared retrospectively the diagnoses established on 2D ultrasound and 3D-CT with the neonatal and/or postmortem work-up, which were used as the gold standard., Results: 2D ultrasound provided the correct diagnosis in only two of the 11 cases. CT yielded the correct diagnosis in eight; in six of these, 2D ultrasound had been inconclusive. 3D-CT was more accurate than was 2D ultrasound in visualizing vertebral anomalies (abnormal shape of the vertebral bodies, abnormal interpedicular distance), pelvic bone malformations (delayed ossification of the pubic bones, abnormal acetabular shape) and enlarged metaphysis or synostoses in long bones. In three cases, neither 2D ultrasound nor CT provided the correct diagnosis., Conclusion: In this series, which included a variety of skeletal dysplasias, 3D-CT had a better diagnostic yield than did 2D ultrasound. Both imaging techniques are useful in the management of fetal dysplasia; 2D ultrasound is a useful screening test and 3D-CT is a valuable complementary diagnostic tool., (Copyright (c) 2007 ISUOG.)

Purpose: The PAX6 gene was first described as a candidate for human aniridia. However, PAX6 expression is not restricted to the eye and it appears to be crucial for brain development. We studied PAX6 mutations in a large spectrum of patients who presented with aniridia phenotypes, Peters' anomaly, and anterior segment malformations associated or not with neurological anomalies., Methods: Patients and related families were ophthalmologically phenotyped, and in some cases neurologically and endocrinologically examined. We screened the PAX6 gene by direct sequencing in three groups of patients: those affected by aniridia; those with diverse ocular manifestations; and those with Peters' anomaly. Two mutations were investigated by generating crystallographic representations of the amino acid changes., Results: Three novel heterozygous mutations affecting three unrelated families were identified: the g.572T>C nucleotide change, located in exon 5, and corresponding to the Leucine 46 Proline amino-acid mutation (L46P); the g.655A>G nucleotide change, located in exon 6, and corresponding to the Serine 74 Glycine amino-acid mutation (S74G); and the nucleotide deletion 579delG del, located in exon 6, which induces a frameshift mutation leading to a stop codon (V48fsX53). The L46P mutation was identified in affected patients presenting bilateral microphthalmia, cataracts, and nystagmus. The S74G mutation was found in a large family that had congenital ocular abnormalities, diverse neurological manifestations, and variable cognitive impairments. The 579delG deletion (V48fsX53) caused in the affected members of the same family bilateral aniridia associated with congenital cataract, foveal hypolasia, and nystagmus. We also detected a novel intronic nucleotide change, IVS2+9G>A (very likely a mutation) in an apparently isolated patient affected by a complex ocular phenotype, characterized primarily by a bilateral microphthalmia. Whether this nucleotide change is indeed pathogenic remains to be demonstrated. Two previously known heterozygous mutations of the PAX6 gene sequence were also detected in patients affected by aniridia: a de novo previously known nucleotide change, g.972C>T (Q179X), in exon 8, leading to a stop codon and a heterozygous g.555C>A (C40X) recurrent nonsense mutation in exon 5. No mutations were found in patients with Peters' anomaly., Conclusions: We identified three mutations associated with aniridia phenotypes (Q179X, C40X, and V48fsX53). The three other mutations reported here cause non-aniridia ocular phenotypes associated in some cases with neurological anomalies. The IVS2+9G>A nucleotide change was detected in a patient with a microphthalmia phenotype. The L46P mutation was detected in a family with microphthalmia, cataract, and nystagmus. This mutation is located in the DNA-binding paired-domain and the crystallographic representations of this mutation show that this mutation may affect the helix-turn-helix motif, and as a consequence the DNA-binding properties of the resulting mutated protein. Ser74 is located in the PAX6 PD linker region, essential for DNA recognition and DNA binding, and the side chain of the Ser74 contributes to DNA recognition by the linker domain through direct contacts. Crystallographic representations show that the S74G mutation results in no side chain and therefore perturbs the DNA-binding properties of PAX6. This study highlights the severity and diversity of the consequences of PAX6 mutations that appeared to result from the complexity of the PAX6 gene structure, and the numerous possibilities for DNA binding. This study emphasizes the fact that neurodevelopmental abnormalities may be caused by PAX6 mutations. The neuro-developmental abnormalities caused by PAX6 mutations are probably still overlooked in the current clinical examinations performed throughout the world in patients affected by PAX6 mutations.

Objective: The aim of this study was to determine whether karyotyping should be performed for every fetal malformation detected in low risk populations., Methods: A karyotype was obtained from 428 fetuses examined over a 10-year period after fetal malformation was diagnosed using obstetrical ultrasound. These fetuses were separated into two groups, one with isolated malformations and the other with multiple malformations. The association between each type of malformation and the result of karyotype was evaluated., Results: Forty-eight chromosomal abnormalities were encountered in 428 fetuses (11.2%). The karyotype was abnormal in 32/343 (9.3%) fetuses with isolated malformations and 16/85 (18.8%) fetuses with multiple malformations (p=0.022). The probability of an abnormal karyotype among the group of isolated malformation depended on the anatomical system involved (p<0.001). Our study demonstrated several isolated malformations without chromosomal abnormality (hydronephrosis with high obstruction, unilateral multicystic dysplastic kidney, gastroschisis, intestinal dilatation, meconium peritonitis, cystic adenomatoid malformation, pulmonary sequestration, tumor, vertebral anomaly)., Conclusion: Each fetus with multiple malformations needs a chromosomal analysis. Within the group of isolated malformations, our study emphasizes that medical maternal history and the type of malformation need to be taken into account before performing a fetal karyotype., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

Purpose: To conduct clinical and genetic studies in a European family with autosomal dominant Stargardt-like macular dystrophy (adSTGD-like MD) and to investigate the functional consequences of a novel ELOVL4 mutation., Methods: Ophthalmic examination and mutation screening by direct sequencing of the ELOVL4 gene was performed in two affected individuals. Wild-type and mutant ELOVL4 genes were expressed as enhanced green fluorescent protein (EGFP) fusion proteins in transient transfection in NIH-3T3 and HEK293 cells. To determine the subcellular localization of ELOVL4, an endoplasmic-reticulum (ER)-specific marker for pDsRed2-ER was cotransfected with ELOVL4 constructs. Transfected cells were viewed by confocal microscopy. Western blot analysis was performed to assess protein expression using an anti-GFP antibody., Results: Affected patients exhibited macular atrophy with surrounding flecks characteristic of adSTGD-like MD. A novel ELOVL4 p.Tyr270X mutation was detected in affected individuals. In cell-transfection studies, wild-type ELOVL4 localized preferentially to the ER. In contrast, the mutant protein appeared to be mislocalized within transfected cells., Conclusions: In a European family with adSTGD-like MD, a novel ELOVL4 mutation was found to underlie the disorder. Transfection studies indicated that, unlike wild-type ELOVL4, the mutant protein does not localize to the ER but rather appears to be sequestered elsewhere in an aggregated pattern in the cytoplasm. Further analysis of the function of normal and mutant ELOVL4 will provide insight into the mechanism of macular degeneration.

Familial porencephaly is a rare condition usually transmitted as an autosomal dominant trait with incomplete penetrance. We describe a new family in which six members across four generations had congenital hemiplegia. Cerebral imaging was performed in three patients and showed porencephaly in all cases. In order to provide effective genetic counseling, three asymptomatic carriers were investigated by cerebral computerized tomography (three patients) and cerebral magnetic resonance imaging (one patient). These investigations failed to show any congenital abnormalities. We conclude that cerebral imaging is unreliable to detect obligate carriers of familial porencephaly., (Copyright 2002 Wiley-Liss, Inc.)

Purpose: To characterize the spectrum of mutations in the OPA1 gene in a large international panel of patients with autosomal dominant optic atrophy (adOA), to improve understanding of the range of functional deficits attributable to sequence variants in this gene, and to assess any genotype-phenotype correlations., Methods: All 28 coding exons of OPA1, intron-exon splice sites, 273 bp 5' to exon 1, and two intronic regions with putative function were screened in 94 apparently unrelated white patients of European origin with adOA by single-strand conformational polymorphism (SSCP)-heteroduplex analysis and direct sequencing. Clinical data were collated, and putative mutations were tested for segregation in the respective families by SSCP analysis or direct sequencing and in 100 control chromosomes. Further characterization of selected splice site mutations was performed by RT-PCR of patient leukocyte RNA. Staining of mitochondria in leukocytes of patients and control subjects was undertaken to assess gross differences in morphology and cellular distribution., Results: Twenty different mutations were detected, of which 14 were novel disease mutations (missense, nonsense, deletion-frameshift, and splice site alterations) and six were known mutations. Mutations were found in 44 (47%) of the 94 families included in the study. Ten new polymorphisms in the OPA1 gene were also identified. Mutations occur throughout the gene, with three clusters emerging: in the mitochondrial leader, in the highly conserved guanosine triphosphate (GTP)-binding domain, and in the -COOH terminus. Examination of leukocyte mitochondria from two unrelated patients with splice site mutations in OPA1 revealed no abnormalities of morphology or cellular distribution when compared with control individuals., Conclusions: This study describes 14 novel mutations in the OPA1 gene in patients with adOA, bringing the total number so far reported to 54. It is likely that many cases of adOA are due to mutations outside the coding region of OPA1 or to large-scale rearrangements. Evaluation of the mutation spectrum indicates more than one pathophysiological mechanism for adOA. Preliminary data suggests that phenotype-genotype correlation is complex, implying a role for other genetic modifying or environmental factors. No evidence was found of pathologic changes in leukocyte mitochondria of patients with adOA.

Mutations in FOXL2, a forkhead transcription factor gene, have recently been shown to cause blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) types I and II, a rare genetic disorder. In BPES type I a complex eyelid malformation is associated with premature ovarian failure (POF), whereas in BPES type II the eyelid defect occurs as an isolated entity. In this study, we describe the identification of novel mutations in the FOXL2 gene in BPES types I and II families, in sporadic BPES patients, and in BPES families where the type could not be established. In 67% of the patients studied, we identified a mutation in the FOXL2 gene. In total, 21 mutations (17 of which are novel) and one microdeletion were identified. Thirteen of these FOXL2 mutations are unique. In this study, we demonstrate that there is a genotype--phenotype correlation for either types of BPES by the finding that mutations predicted to result in a truncated protein either lacking or containing the forkhead domain lead to BPES type I. In contrast, duplications within or downstream of the forkhead domain, and a frameshift downstream of them, all predicted to result in an extended protein, cause BPES type II. In addition, in 30 unrelated patients with isolated POF no causal mutations were identified in FOXL2. Our study provides further evidence that FOXL2 haploinsufficiency may cause BPES types I and II by the effect of a null allele and a hypomorphic allele, respectively. Furthermore, we propose that in a fraction of the BPES patients the genetic defect does not reside within the coding region of the FOXL2 gene and may be caused by a position effect.

Choreoathetosis is a major clinical feature in only a small number of hereditary neurological disorders. We define a new X-linked syndrome with a unique clinical picture characterized by mild mental retardation, choreoathetosis, and abnormal behavior. We mapped the disease in a four-generation pedigree to chromosome Xp11 by linkage analysis and defined a candidate region containing a number of genes possibly involved in neuronal signaling, including a potassium channel gene and a neuronal G protein-coupled receptor.

Spinocerebellar ataxia type 7 (SCA7), in which the degenerative process also affect the retina, belongs to the category of the autosomal dominant cerebellar ataxia type II (ADCA II). We have described the neuropathology of this condition [Martin JJ, Van Regemorter N, Krols L, Brucher JM, de Barsy T, Szliwowski H, et al. On an autosomal dominant form of retino-cerebellar degeneration: an autopsy study of five patients in one family. Acta Neuropathol (Berl) 1994;88:277-286] in a very large Belgian family (CA-1). We have observed anticipation in the age of onset with increasing severity of the symptoms in consecutive generations. The SCA7 gene was mapped to chromosome 3p12-13 [David G, Abbas N, Stevanin G, Dürr A, Yvert G, Cancel G, et al. Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion. Nat Genet 1997;17:65-70; Del-Favero J, Krols L, Michalik A, Theuns J, Löfgren A, Goossens D, et al. Molecular genetic analysis of autosomal dominant cerebellar ataxia with retinal degeneration (ADCA type II) caused by CAG triplet repeat expansion. Hum Mol Genet 1998;7:177-186], and the gene identified. SCA7 is a new gene of unknown function that contains an expansion of CAG repeats in SCA7 patients. During the procedure of positional cloning, we examined 26 patients belonging to the CA-1 family and realized, in some of them, an ophthalmologic examination and neuro-imaging of the brain. This allowed us to differentiate four groups: (1) asymptomatic young carriers with 38 to 43 CAG repeats; (2) mildly symptomatic, older patients with 38-41 CAG repeats; (3) patients with the full-blown picture of SCA7 and age of onset during adolescence, with 54-55 CAG repeats; (4) children with early onset and rapid fatal course of the disease who had over 55 CAG repeats. We were able to draw correlations between clinical phenotype, age at onset and CAG repeat number and to make predictions, to some extent, as to the clinical course of the disease in new patients.

Deletions of the short arm of chromosome 6 are relatively rare, the main features being developmental delay, craniofacial malformations, hypotonia, and defects of the heart and kidney, with hydrocephalus and eye abnormalities occurring in some instances. We present the molecular cytogenetic investigation of six cases with 6p deletions and two cases with unbalanced translocations resulting in monosomy of the distal part of 6p. The breakpoints of the deletions have been determined accurately by using 55 well-mapped probes and fluorescence in situ hybridization (FISH). The cases can be grouped into two distinct categories: interstitial deletions within the 6p22-p24 segment and terminal deletions within the 6p24-pter segment. Characteristics correlating with specific regions are: short neck, clinodactyly or syndactyly, brain, heart and kidney defects with deletions within 6p23-p24; and corneal opacities/iris coloboma/Rieger anomaly, hypertelorism and deafness with deletions of 6p25. The two cases with unbalanced translocations presented with a Larsen-like syndrome including some characteristics of the 6p deletion syndrome, which can be explained by the deletion of 6p25. Such investigation of cytogenetic abnormalities of 6p using FISH techniques and a defined set of probes will allow a direct comparison of reported cases and enable more accurate diagnosis as well as prognosis in patients with 6p deletions.

Trichothiodystrophy was diagnosed in a 3-year-old male presenting with speech delay, brittle hair, chronic neutropenia, and a history of febrile convulsions. Cranial magnetic resonance imaging revealed a focal subcortical and periventricular gray matter heterotopia. An acute encephalopathy with status epilepticus and coma occurred when he was 4 years of age during an upper respiratory tract infection. Magnetic resonance imaging revealed multifocal T2-weighted hypersignal lesions involving mainly the thalami, hippocampi, midbrain, and pons. Analysis of cerebrospinal fluid revealed hyperproteinorachia without pleocytosis. Results of an extensive metabolic evaluation of this acute brain injury, resembling the syndrome of acute necrotizing encephalopathy of childhood described in Japan, were negative. Focal neuronal migration disorder and acute encephalopathy with symmetric thalamic involvement are newly described neurologic manifestations of syndromes with trichothiodystrophy, which suggests that these conditions may have a common genetic background.

The 3 affected children from 2 different wedlocks of the mother have been previously described (11). Search by FISH analysis in the mother revealed she is a carrier of balanced translocation of clear terminal G bands of equal sizes of the long arms of chromosomes 10 and 14. Chromosomal slides of the last child (Patient 3) could be analysed by fish and revealed that he did inherit the derivative chromosome 10. He had a partial trisomy 14 and a partial deletion of the long arm of chromosome 10. The clinical pictures correspondence to the possibly abnormal karyotypes will be discussed.

We have previously mapped the gene for autosomal dominant cerebellar ataxia type II (ADCAII) to chromosome 3p12-p21.1 in a region of 33 cM by using four families of different geographic origin. In this study, we analysed the families with nine additional simple tandem repeat markers located in the ADCAII candidate region. An extensive clinical evaluation was also performed in the Belgian family CA-1 on two probably affected and seven at-risk individuals by means of ophthalmological examination and magnetic resonance imaging. Based on informative recombinants, we were able to reduce the ADCAII candidate region to the 12-cM region between D3S1300 and D3S1285. Furthermore, haplotype analysis among the families suggested that the most likely location of the ADCAII gene is within the 6.2-cM interval between D3S3698 and D3S1285. Because of the documented anticipation in ADCAII families, we also analysed family CA-1 with six polymorphic triplet repeat markers located on chromosome 3. None of these markers showed expanded alleles.

A candidate gene for Branchio-Oto-Renal (BOR) syndrome was identified at chromosome 8q13.3 by positional cloning and shown to underlie the disease. This gene is a human homologue of the Drosophila eyes absent gene (eya), and was therefore called EYA1. A highly conserved 271-amino acid C-terminal region was also found in the products of two other human genes (EYA2 and EYA3), demonstrating the existence of a novel gene family. The expression pattern of the murine EYA1 orthologue, Eya1, suggests a role in the development of all components of the inner ear, from the emergence of the otic placode. In the developing kidney, the expression pattern is indicative of a role for Eya1 in the metanephric cells surrounding the 'just-divided' ureteric branches.

Deletions of the short arm of chromosome 6 are relatively rare, only 16 cases having been described in the literature so far. Here we present a detailed investigation by fluorescence in situ hybridisation of two further cases with different but overlapping interstitial deletions involving 6p22, 6p23 and 6p24. The main features involved are craniofacial malformations, heart and kidney defects, mental retardation/developmental delay, hypotonia and hydrocephalus. By using 36 yeast artificial chromosome and cosmid clones from a contig covering 6p22.3-6p25 and other probes with defined cytogenetic locations within 6p21-6p22 we have precisely localised the breakpoints involved in each of the cases, estimated the sizes of the deleted regions and defined the region that is hemizygously deleted in both cases.

We have characterised a point mutation causing the substitution of serine for glycine at position 661 of the alpha1(I) chain of type I collagen in a child with a severe form of osteogenesis imperfecta. An identical glycine substitution in the alpha2(I) chain was previously detected in a woman with post-menopausal osteoporosis. Two of her sons were heterozygous for the mutation and the third son was homozygous as a result of uniparental isodisomy. Biochemical profiles of the type I collagen heterotrimers were studied in each of the patients and compared with a control. Medium and cell-layer collagens were overmodified in all patients. Overmodification was obvious in the patient with the alpha 1(I) mutation but mild in the patients with the alpha 2(I) mutation, being slightly less evident in the heterozygote than in the homozygote. Investigation of the melting curves of the mutant collagen trimers in all three patients showed the same slight decrease in thermal stability and, hence, a lack of correlation with phenotypic severity. In contrast, the degree of overmodification of the collagen alpha chains was correlated with the phenotypic severity. The clinical observations in these patients illustrate the possibly predominant role of mutations in the collagen alpha1(I) chains over the same mutations in the alpha2(I) chains in determining the clinical outcome.

Metachromatic leukodystrophy (MLD) is an autosomal recessive disease of myelin metabolism caused by a deficiency in the lysosomal enzyme arylsulphatase A (ARSA). We have identified a new mutation in exon 4 of the ARSA gene of two unrelated Belgian patients with late-infantile MLD. The mutation predicts an aspartic acid-to-histidine substitution at position 255 in arylsulphatase A (D255H), in a highly conserved region among sulphatases. Transient expression of the mutation in COS cells did not show an increase in ARSA activity. Both patients were compound heterozygotes carrying the frequent splice site mutation in intron 2 (459 + IG -->A) on the other allele.

We report an association of trigonocephaly and thumb hypoplasia in a 6.5-year-old boy, diagnosed as Baller-Gerold syndrome. In addition to craniosynostosis and radial limb defect, which are constant in this syndrome, our patient presents two unusual features: the first is an epidermal nevus and the second is an agenesis of the middle portion of corpus callosum. This unique type of callosal agenesis in the context of a polymalformative disorder supports the hypothesis that partial agenesis of corpus callosum may be due to an event occurring before the 12th week gestation with continued development of the midline structures.

Between October 1985 and December 1993, 421 patients underwent fetal blood sampling for rapid karyotyping (426 samplings, 5 twin pregnancies). The aim of the study was to evaluate cordocentesis in terms of results, complications and additional information in this specific indication especially in case of abnormal pregnancy sonogram. The fetal loss rate possibly related to fetal blood sampling was 1.9%. Abnormal sonograms represented 91% of rapid karyotype indications. Chromosomal abnormalities were found in 9.5% of abnormal sonograms and in 16.9% of fetal structural anomalies (37 cases). The commonest chromosomal abnormality was trisomy 21 (11 cases). In conclusion, cordocentesis is a safe and reliable method for rapid karyotyping although it is associated with more risks than in other indications (congenital infections). The high rate of chromosomal abnormalities pleaded for ultrasonographic screening in a population usually not investigated by cytogenetic studies.

We have studied a girl with multiple congenital anomalies, growth and mental deficiency, characteristic facial anomalies, cataracts, cerebellar atrophy, and severe hypocholesterolemia. Death occurred at age 7 years. After excluding several syndromes, i.e., peroxisomal disorders, mevalonic acidaemia, and Marinesco-Sjögren syndrome, it is concluded that this girl had severe Smith-Lemli-Opitz Syndrome (SLOS) with exceptionally long survival. This diagnosis was confirmed through assay of 7-dehydrocholesterol in cultured fibroblasts.

Foetal blood sampling was performed at 35 weeks of gestation due to abnormal foetal ultrasound findings. There was apparent monosomy 21 (45,XX,-21) in all mitoses analyzed. The infant died at 37 weeks during delivery. Examination disclosed facial anomalies, clubfeet, hypoplasia of the left urogenital tract, agenesis of corpus callosum, ventricular dilatation, and heterotopias. Reevaluation of the karyotype showed an unbalanced translocation t(1;21) (q44;q22.11) which resulted from a maternal balanced translocation. These findings were confirmed by fluorescence in situ hybridization and molecular studies with chromosome 21 specific markers. The latter showed a proximal deletion of the maternally derived chromosome 21 including all loci from centromere down to the D21S210 locus. This case illustrates the need for complementary cytogenetic and molecular investigations in cases of apparent monosomy 21.

We describe a family with an autosomal dominant form of retinal-cerebellar atrophy. There is an extreme variability in age of onset and severity of the clinical symptoms: some patients remain nearly asymptomatic throughout their entire life; others develop severe retinal and cerebellar symptoms after the age of 35 years; others suffer from a severe disorder with onset in adolescence and death during the third decade of life; in others the onset is in early childhood with prevalence of cerebellar symptoms. There is neither dementia nor epilepsy in any of the patients. Four out of five autopsies showed a severe retinal atrophy, and all five autopsies were also characterized by (1) a cerebellar atrophy affecting the spinocerebellar and olivocerebellar tracts, the cerebellar cortex and the efferent cerebellar pathways, (2) an involvement of the pyramidal pathways and of the motor neurons of brain stem and spinal cord, and (3) an atrophy of the subthalamic nucleus and to a much lesser extent of the pallidum, with also some damage to the substantia nigra. The posterior columns are much less affected except in one patient. In this family, we have excluded linkage with the two loci for spinocerebellar ataxia, i.e., SCA1 on chromosome 6p and SCA2 on chromosome 12q as well as with the locus for Machado-Joseph disease (MJD) on chromosome 14q. A genome-wide search is currently being performed to detect the disease locus responsible.

The birth of a male baby was induced at 32 weeks. In utero, the child presented, inter alia, signs of hydrops, hepatosplenomegaly and anaemia. Two in utero transfusions for correction of the anaemia were performed at 28 and 29 weeks, respectively. The baby rapidly presented respiratory distress with mixed acidosis. Three hours after birth, pink urine was excreted. Signs of icterus necessitated phototherapy, after which photosensitivity occurred. Erythrocytes were fluorescent under long-wavelength UV light. The baby died 24 hours after birth, displaying severe acidosis, a diffuse haemorrhagic syndrome, and repeated brady-cardia which did not respond to isoprenaline. The analysis of porphyrins in urine, blood and faeces of the baby gave the following results: 1) uroporphyrin (I and III isomeric series) was increased in urine and faeces, with traces in erythrocytes and plasma; 2) heptacarboxyporphyrin I was found mainly in urine and much less in erythrocytes, plasma and faeces; 3) coproporphyrin I was increased in urine, erythrocytes, plasma and faeces, and 4) 5-aminolaevulinic acid and porphobilinogen in urine and plasma were within the reference ranges. Determination of the enzymes of haem biosynthesis in erythrocytes and lymphocytes showed that both parents possessed only 50% of the normal activity of cosynthase. A previously described point mutation in codon 73 was observed in one parent. Fatal cases of neonatal Günther's disease are extremely rare and such an observation, according to our knowledge, is probably one of the first described.

In this report, we describe three sibs presenting an identical malformation syndrome i.e.: acrocephaly, brachydactyly, prominent metopic ridge, broad depressed nasal bridge, narrow maxillae, obesity and normal intelligence. We discuss the relationship between this combination of clinical signs and symptoms most compatible with the diagnosis of Summitt syndrome and the Carpenter syndrome.

The authors describe a girl with retarded growth, renal malformations, retinal coloboma and hypoplasia of the thumbs like reported in the acro-renal-ocular syndrome. In addition, they found defects of the ribs and spine and cleft palate, not previously described in this syndrome. The was noted to have minor anomalies of the hands, which might represent a mild manifestation of this autosomal dominant syndrome.

Two unrelated children presented with similar clinical features (facial dysmorphism and multiple joint dislocations) suggesting the diagnosis of Larsen syndrome. Both carried an inherited unbalanced translocation resulting in partial trisomy 1q and partial monosomy 6p. Analysis of skin collagen from one of the probands disclosed a decreased alpha 1/alpha 2 chain ratio of collagen type I, increased thermal stability and increased hydroxylation of proline and lysine. The present findings suggest that, as a result of the chromosome rearrangements, both patients have a mutation on a gene involved in collagen production, located either on chromosome 1q or, more probably, on 6p. It is furthermore suggested that other cases of Larsen syndrome are the result of a similar mutation.

Partial androgen insensitivity syndrome (PAIS) is an X-linked disorder resulting from defects in the intracellular androgen receptor (AR). The cloning of the AR cDNA has provided the molecular tools to identify gene abnormalities. Gene deletions being the exception in PAIS, prenatal diagnosis of PAIS resulting from a single base mutation in high risk families is not practical unless the mutation is already known. Brown et al. (1989) reported that 10% of normal X chromosomes present a Hind III 6.7/3.5 kb polymorphism. In this study, we report the association of the Hind II polymorphism in a woman whose son has a PAIS associated with a very low androgen receptor concentration: we differentiated the two maternal X chromosomes and characterized the affected allele. These data demonstrate that the presence of Hind III polymorphic fragments could be used in prenatal diagnosis of androgen insensitivity syndrome in high risk families.

We report a newborn with some manifestations of chondrodysplasia punctata. Additional abnormalities were hydrocephalus, bilateral syndactyly of the fourth and fifth fingers and toes, absence of the middle phalanx of all toes, hypoplasia of the second and third phalanges of all fingers and cryptorchidism. This observation suggests that we are possibly dealing with a rare male case of X-linked dominant chondrodysplasia punctata.

Alphafetoprotein (AFP) and concanavalin A non-reactive alphafetoprotein determination and the acetylcholinesterase (AchE) qualitative test have been performed on amniotic fluid samples from 33 normal pregnancies, 44 pregnancies with fetal malformations and 8 normal pregnancies with elevated amniotic fluid alphafetoprotein (3 false positive AFP results, 5 contaminations with fetal blood). The validities of these three tests in detecting abnormal pregnancies are compared. The usefulness of the existing complementary tests in the detection of neural tube defects in a low neural tube defect incidence area is discussed. Risk figures for open spina bifida according to the prior risk situation and the results of maternal serum AFP, amniotic fluid AFP, AchE qualitative test and ultrasound examination have been calculated.

We report on two fetuses with a lethal form of multiple-pterygium syndrome born to first cousins. The two pregnancies aborted spontaneously in the 2nd trimester, the fetuses, 10-11 weeks of embryonic age, showing multiple pterygia and multiple cartilaginous fusions. One had cleft lip and palate. In both cases the microscopic anatomy of the placenta showed villi with scalloped border and intravillous trophoblastic invaginations.

Two siblings affected with partial trisomy 3p resulting from a maternal balanced translocation 46,XXt(3;10) (p21;q26) are reported. The clinical features of this syndrome are reviewed. The necropsy findings of the two siblings are discussed.

Severe osteopetrosis was diagnosed in utero in two successive pregnancies resulting from an intermarriage. Hydrocephaly and skeletal hyperdensity were detected at 18 weeks of gestation, and fractures at 24 weeks. We report on extensive ultrasound, radiological, and pathological findings, including those on brain and bone. The markedly reduced number of osteoclasts observed in these sibs and the very early fetal involvement suggest that this form of osteopetrosis might represent a new entity: autosomal recessive lethal osteopetrosis.

Three related new cases with almost complete trisomy 11p due to paternal balanced translocation 46, XY, t(7; 11) (q36.1; p11.1) are reported. The proband (Case 1) was a malformed stillborn with exomphalos, case 2 was diagnosed in the first trimester by direct chromosome preparations from chorionic villi, and confirmed on fetal products after termination of pregnancy. Case 3, a cousin to cases 1 and 2, was a 29-weeks-old fetus with omphalocele discovered at ultrasound. Literature reports of trisomy 11p are reviewed with regard to those new cases, and the possible relationship of this chromosome imbalance with the Beckwith-Wiedemann Syndrome is discussed.

S-100 protein, which is found essentially in the astrocytes of the nervous system, was assayed in amniotic fluids by Particle Counting ImmunoAssay. It was present in 19 cases of anencephaly out of 26, in 1 case of open spina bifida out of 5 and in each of the 4 cases of fetal death, whereas it was not detected in the 48 control amniotic fluids collected between the 16th and the 35th week of gestation. Thirty-one amniotic fluids from fetuses with other congenital malformations were devoid of detectable S-100. The presence of S-100 in amniotic fluid of anencephalic fetuses can presumably be considered as a biological sign of necrosis of the exencephalic brain and seems specific to damage of the central nervous system accompanied by neural tube defect.

Recurrence risks for the generalised single locus model and for the multifactorial model have been derived and compared. First the areas of overlap for the two models were determined and sets of parameters chosen to represent these overlap areas. Certain sets of parameters for the single locus model give recurrence risks in sibships similar to these for multifactorial inheritance. Other sets (representing very low penetrant dominant genes) give markedly lower risks. Similar results hold for more complex family histories. Empiric risks for families with two or more affected individuals are needed so as to indicate the trend of the increase in risk which could then be extrapolated to other families and be used in genetic counseling.

Late amniocenteses (greater than 20 weeks' gestation) were performed in 114 pregnancies with no a priori genetic risk, but referred because of abnormal clinical and/or ultrasound findings suggesting fetal malformations. Reasons for referral included polyhydramnios (51 cases), oligohydramnios (15 cases), fetal growth retardation (FGR) (16 cases) and abnormal fetal ultrasound findings excluding anencephaly (32 cases). In 42 of these cases, referral was motivated by a combination of the above abnormal findings. When polyhydramnios was the sole anomaly (25 cases), 5 fetuses were malformed (20%), abnormal fetal karyotype and/or elevated amniotic fluid alphafetoprotein (AFP) were demonstrated in 2 cases. Oligohydramnios was the sole anomaly in one case; the infant died of prematurity. Fetal growth retardation was the sole anomaly in 14 cases, 11 otherwise normal newborns were small for date, 2 died at birth and 1 was malformed (1/14, 7%). In this group all fetal karyotypes were normal and in 2 cases amniotic fluid AFP were increased. In the 32 pregnancies without abnormal amniotic fluid volume and/or FGR and with fetal malformation(s) suggested by ultrasound, all malformations except one (ovarian cyst possibly ruptured during birth) were confirmed at birth, amniotic fluid AFP was elevated, and/or karyotype was abnormal in 6 cases. In 42 pregnancies where more than one alarm sign was present, abnormal karyotype and/or elevated amniotic fluid AFP level were recorded in 21 of the 39 cases where amniocentesis was performed, 33 fetuses were malformed (79%) and 13 died in the perinatal period (31%). The high incidence of abnormal results of amniocentesis found in this survey of pathological pregnancies, particularly in those with multiple alarm signs, emphasizes the need for amniocentesis in these situations.