Cobblestone-like brain dysgenesis and altered glycosylation in congenital cutis laxa, Debre type.

OBJECTIVE: To delineate a new syndrome of brain dysgenesis and cutis laxa based on the description of 11 patients belonging to nine unrelated families recruited through an international collaboration effort. METHODS: Careful clinical assessment of patients from birth to the age of 23 years with follow-up studies ranging from 3 to 20 years. Biochemical studies of serum proteins glycosylation by isoelectric focusing and capillary zone electrophoresis were performed in 10 patients. Brain MRI studies using conventional methods were analyzed in eight patients. RESULTS: An expanded clinical spectrum of a syndrome comprising facial dysmorphia (enlarged anterior fontanelles, downward slant of palpebral fissures, prominent root of the nose), a connective tissue disorder (inguinal hernia, hip dislocation, high myopia), and neurologic impairment was defined. Early developmental delay was followed by onset of generalized seizures by the end of the first decade and a subsequent neurodegenerative course. A defect of N- or N- plus O-glycosylation of serum transferrins and ApoCIII was observed in 10 patients. An unusual cobblestone-like cortical malformation over the frontal and parietal regions was seen in eight patients and cerebellar abnormalities, including two patients with Dandy-Walker malformation, were observed in three patients. CONCLUSIONS: Our results suggest that autosomal recessive cutis laxa, Debré type, initially considered a dermatologic syndrome, is a multisystemic disorder with cobblestone-like brain dysgenesis manifesting as developmental delay and an epileptic neurodegenerative syndrome. It might represent a metabolic cause of Dandy-Walker malformation. It is associated with a deficient N- and-O glycosylation of proteins and shares many similarities with muscle-eye-brain syndromes.

Loss of desmoplakin isoform I causes early onset cardiomyopathy and heart failure in a Naxos-like syndrome.

BACKGROUND: Desmosomes are cellular junctions important for intercellular adhesion and anchoring the intermediate filament (IF) cytoskeleton to the cell membrane. Desmoplakin (DSP) is the most abundant desmosomal protein with 2 isoforms produced by alternative splicing. METHODS: We describe a patient with a recessively inherited arrhythmogenic dilated cardiomyopathy with left and right ventricular involvement, epidermolytic palmoplantar keratoderma, and woolly hair. The patient showed a severe heart phenotype with an early onset and rapid progression to heart failure at 4 years of age. RESULTS: A homozygous nonsense mutation, R1267X, was found in exon 23 of the desmoplakin gene, which results in an isoform specific truncation of the larger DSPI isoform. The loss of most of the DSPI specific rod domain and C-terminal area was confirmed by Western blotting and immunofluorescence. We further showed that the truncated DSPI transcript is unstable, leading to a loss of DSPI. DSPI is reported to be an obligate constituent of desmosomes and the only isoform present in cardiac tissue. To address this, we reviewed the expression of DSP isoforms in the heart. Our data suggest that DSPI is the major cardiac isoform but we also show that specific compartments of the heart have detectable DSPII expression. CONCLUSIONS: This is the first description of a phenotype caused by a mutation affecting only one DSP isoform. Our findings emphasise the importance of desmoplakin and desmosomes in epidermal and cardiac function and additionally highlight the possibility that the different isoforms of desmoplakin may have distinct functional properties within the desmosome.

A new syndrome, congenital extraocular muscle fibrosis with ulnar hand anomalies, maps to chromosome 21qter.

BACKGROUND: Congenital fibrosis of the extraocular muscles (CFEOM) is a heterogeneous group of disorders that may be associated with other anomalies. The association of a CFEOM syndrome with ulnar hand abnormalities (CFEOM/U) has not been reported to date. OBJECTIVE: To describe a new autosomal recessive syndrome of CFEOM and ulnar hand abnormalities, and localise the disease causing gene. METHODS: Clinical evaluation of the affected members and positional mapping. RESULTS: Six affected patients with CFEOM/U (aged 2 to 29 years) from a large consanguineous Turkish family were studied. Ophthalmological involvement was characterised by non-progressive restrictive ophthalmoplegia with blepharoptosis of the right eye. The postaxial oligodactyly/oligosyndactyly of the hands was more severe on the right side. A genome-wide scan established linkage of this new autosomal recessive syndrome to a locus on chromosome 21qter. The multipoint LOD score was 4.53 at microsatellite marker D21S1259, and fine mapping defined a approximately 1.5 Mb critical region between microsatellite marker D21S1897 and the telomere of the long arm. CONCLUSIONS: CFEOM/U maps to a 1.5 Mb region at chromosome 21qter. Future identification of the disease causing gene may provide insights into the development of the extraocular muscles and brain stem alpha motor neurones, as well as anteroposterior limb development.

The results of cytogenetic analysis with regard to intracytoplasmic sperm injection in males, females and fetuses.

OBJECTIVES: To determine the incidence of chromosome abnormalities among couples for whom intracytoplasmic sperm injection (ICSI) treatment was indicated and fetuses conceived through the ICSI procedure. METHODS: All cytogenetic results were evaluated retrospectively. Patients undergoing ICSI (n = 508) were classified according to the referring indications as: (1) males with severe infertility (87 azoospermia and 34 oligoasthenoteratozoospermia, OAT), (2) prior to ICSI (56 males and 61 females), and (3) following an unsuccessful ICSI procedure (132 males and 138 females). Fetuses conceived through ICSI (n = 475) were also classified into 4 groups according to the additional risk factors for chromosome abnormalities: ICSI (n = 185), ICSI + advanced maternal age (AMA, n = 215), ICSI + positive triple test result (TT, n = 50), and ICSI + abnormal ultrasound findings (USG, n = 25). RESULTS: An abnormal karyotype was found in 31.03% of males with azoospermia and 14.71% of males with OAT, in 3.57% of males and 1.64% of females in the group prior to ICSI, and in 5.30 and 5.07%, respectively, in the group following unsuccessful ICSI treatment. Gonosomal aneuploidies were predominant in males with azoospermia and autosomal rearrangements in males with OAT, while low-level sex chromosome mosaicism was found in females. The overall frequency of chromosome abnormalities in fetuses was 4.42% and varied in the different groups from 1.62% in ICSI, 2.79% in ICSI + AMA, 10.0% in ICSI + TT to 28.0% in ICSI + USG. The frequencies of the different types of chromosome abnormalities were as follows: balanced 1.05%, unbalanced 3.37%, familial 0.84%, de novo 3.37%, autosomal 3.58%, gonosomal 0.84%, numerical 1.89%, structural abnormalities 2.53%, and mosaicism 1.26%. CONCLUSION: Our results indicate that cytogenetic investigations of the ICSI parents and fetuses are essential for the families, genetic counselors and also reproductive centers. In fetal karyotyping, de novo structural chromosome abnormalities and mosaicism should be taken into consideration.

A novel semiquantitative polymerase chain reaction/enzyme digestion-based method for detection of large scale deletions/conversions of the CYP21 gene and mutation screening in Turkish families with 21-hydroxylase deficiency.

21-Hydroxylase deficiency is a recessively inherited disorder resulting from mutations in the CYP21 gene. The CYP21 gene is located along with the CYP21P pseudogene in the human leukocyte antigen major histocompatibility complex region on chromosome 6. Molecular diagnosis is difficult due to the 98% similarity of CYP21 and CYP21P genes and the fact that almost all frequently reported mutations reside on the pseudogene. Allele-specific PCR for the 8 most frequently reported point mutations was performed in 31 Turkish families with at least a single 21-hydroxylase-deficient individual. The allele frequencies of the point mutations were as follows: P30L, 0%; IVS2 (AS,A/C-G,-13), 22.5%; G110delta8nt, 3.2%; I172N, 11.4%; exon 6 cluster (I236N, V237E, M239K), 3.2%; V281L, 0%; Q318X, 8%; and R356W, 9.6%. Large deletions and gene conversions were detected by Southern blot analysis, and the allele frequencies were 9.6% and 22.5%, respectively. Sequence analysis of the gene, performed on patients with only 1 mutated allele, revealed 2 missense mutations (R339H and P435S). A novel semiquantitative PCR/enzyme digestion-based method for the detection of large scale deletions/conversions of the gene was developed for routine diagnostic purposes, and its accuracy was shown by comparison with the results of Southern blot analysis.

Molecular analyses of the HGO gene mutations in Turkish alkaptonuria patients suggest that the R58fs mutation originated from central Asia and was spread throughout Europe and Anatolia by human migrations.

Alkaptonuria (AKU) is a rare metabolic disorder of phenylalanine catabolism that is inherited as an autosomal recessive trait. AKU is caused by loss-of-function mutations in the homogentisate 1,2-dioxygenase (HGO) gene. The deficiency of homogentisate 1,2-dioxygenase activity causes homogentisic aciduria, ochronosis and arthritis. We present the first molecular study of the HGO gene in Turkish AKU patients. Seven unrelated AKU families from different regions in Turkey were analysed. Patients in three families were homozygous for the R58fs mutation; another three families were homozygous for the R225H mutation; and one family was homozygous for the G270R mutation. Analysis of nine intragenic HGO polymorphisms showed that the R58fs, R225H and G270R Turkish AKU mutations are associated with specific HGO haplotypes. The comparison with previously reported haplotypes associated with these mutations from other populations revealed that the R225H is a recurrent mutation in Turkey, whereas G270R most likely has a Slovak origin. Most interestingly, these analyses showed that the Turkish R58fs mutation shares an HGO haplotype with the R58fs mutation found in Finland, Slovakia and India, suggesting that R58fs is an old AKU mutation that probably originated in central Asia and spread throughout Europe and Anatolia during human migrations.

Frequencies of gap- and tight-junction mutations in Turkish families with autosomal-recessive non-syndromic hearing loss.

Mutations in genes encoding gap- and tight-junction proteins have been shown to cause distinct forms of hearing loss. We have now determined the GJB2[connexin 26 (Cx26)] mutation spectrum in 60 index patients from mostly large Turkish families with autosomal-recessive inherited non-syndromic sensorineural hearing loss (NSSHL). GJB2 mutations were found in 31.7% of the families, and the GJB2-35delG mutation accounted for 73.6% of all GJB2 mutations. The carrier frequency of GJB2-35delG in the normal Turkish population was found to be 1.17% (five in 429). In addition to the described W24X, 233delC, 120delE and R127H mutations, we also identified a novel mutation, Q80R, in the GJB2 gene. Interestingly, the Q80R allele was inherited on the same haplotype as V27I and E114G polymorphisms. As little is known about the mutation frequencies of most other recently identified gap- and tight-junction genes as a cause for hearing loss, we further screened our patients for mutations in GJB3 (Cx31), GJA1 (Cx43), DeltaGJB6-D13S1830 (Cx30) and the gene encoding the tight-junction protein, claudin 14 (CLDN14). Several novel polymorphisms, but no disease-associated mutations, were identified in the CLND14 and GJA1 genes, and we were unable to detect the DeltaGJB6-D13S1830 deletion. A novel putative mutation, P223T, was found in the GJB3 gene in heterozygous form in a family with two affected children. Our data shows that the frequency of GJB2 mutations in Turkish patients with autosomal-recessive NSSHL and the carrier rate of the GJB2-35delG mutation in the Turkish population, is much lower than described for other Mediterranean countries. Furthermore, mutations in other gap- and tight-junction proteins are not a frequent cause of hearing loss in Turkey.

A familial Xp+ chromosome detected during fetal karyotyping, which is associated with short stature in four generations of a Turkish family.

The short-stature homeobox-containing gene (SHOX) on chromosome Xp22.3 was recently identified as an important determinant of the stature phenotype. Deletions of the SHOX gene, some of them due to structural chromosome abnormalities, have been described in patients with idiopathic short stature and Leri-Weill syndrome. Additionally, haploinsufficiency of SHOX is a main cause for short stature seen in patients with Turner syndrome. Here we report an unusual X-chromosome abnormality, which was detected during a fetal karyotyping performed because of a previous child with Down syndrome. GTG banding demonstrated an extra chromosome segment on the terminal part of the short arm of chromosome X in the index case (karyotype: 46,X,Xp+). The same chromosomal abnormality was found in the mother and the maternal grandmother. All carriers of this chromosomal abnormality presented with short stature but no other associated symptoms. Whole chromosome painting of X revealed a homogeneous painting of the abnormal X chromosome indicating that no other chromosome was involved. Additional FISH studies with probe DXS1140 (Kallmann probe at Xp22.3), Quint-Essential X-Specific DNA (DMD probe at Xp21.2), XIST (at Xq13.2), and Tel Xq/Yq were performed, and no abnormality was observed in the intensities or the localizations of the probes signals. However, applying a specific SHOX gene probe (derived from cosmid LLNONO3M34F5) showed a loss of signal on the derivative X chromosome. Our results show that the Xp+ generation led to a deletion of the complete SHOX gene and caused short stature in the presented family.

The novel R75Q mutation in the GJB2 gene causes autosomal dominant hearing loss and palmoplantar keratoderma in a Turkish family.

Dominant mutations in the GJB2 gene encoding connexin 26 (Cx26) can cause non-syndromic hearing impairment alone or in association with palmoplantar keratoderma (PPK). We have identified the novel G224A (R75Q) mutation in the GJB2 gene in a four-generation family from Turkey with autosomal dominant inherited hearing impairment and PPK. The age of onset and progression of hearing loss were found to be variable among affected family members, but all of them had more severe impairment at higher hearing frequencies. Interestingly, the novel R75Q mutation affects the same amino acid residue as described recently in a small family (R75W) with profound prelingual hearing loss and PPK. However, the R75W mutation was also observed in a control individual without PPK and unknown hearing status. Therefore, the nature of the R75W mutation remains ambiguous. Our molecular findings provide further evidence for the importance of the conserved R75 in Cx26 for the physiological function of the inner ear and the epidermal cells of the skin.

Prenatal prediction of childhood-onset spinal muscular atrophy (SMA) in Turkish families.

Childhood-onset spinal muscular atrophy (SMA) is one of the most common neurodegenerative genetic disorders. SMN1 is the SMA-determining gene deleted or mutated in the majority of SMA cases. There is no effective cure or treatment for this disease yet. Thus, the availability of prenatal testing is important. Here we report prenatal prediction for 68 fetuses in 63 Turkish SMA families using direct deletion analysis of the SMN1 gene by restriction digestion. The genotype of the index case was known in 40 families (Group A) but unknown in the remaining 23 families (Group B). A total of ten fetuses were predicted to be affected. Eight of these fetuses were derived from Group A and two of these fetuses were from Group B families. Two fetuses from the same family in Group A had the SMNhyb1 gene in addition to homozygous deletion of the NAIP gene. One fetus from Group A was homozygously deleted for only exon 8 of the SMN2 gene, and further analysis showed the presence of both the SMN1 and SMNhyb1 genes but not the SMN2 gene. In addition, one carrier with a homozygous deletion of only exon 8 of the SMN1 gene was detected to have a SMNhyb2 gene, which was also found in the fetus. To our knowledge, these are the first prenatal cases with SMNhyb genes. Follow-up studies demonstrated that the prenatal predictions and the phenotype of the fetuses correlated well in 33 type I pregnancies demonstrating that a careful molecular analysis of the SMN genes is very useful in predicting the phenotype of the fetus in families at risk for SMA.

CADASIL syndrome in a large Turkish kindred caused by the R90C mutation in the Notch3 receptor.

Mutations in the Notch3 gene are the cause of the autosomal dominant disorder CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). The CADASIL is an adult-onset neurologic disorder (average age of onset is 45 years) characterized by recurrent strokes and dementia. Clinical features combined with cerebral magnetic resonance imaging (MRI), showing a diffuse leukoencephalopathy with subcortical infarcts in the basal ganglia and white matter, are highly contributive to the diagnosis. We present a Turkish family with CADASIL, in which 12 individuals in four generations were affected showing the typical clinical features of recurrent strokes. Mutation analysis of the Notch3 receptor gene identified the recently described R90C mutation in the N-terminal part of the gene in affected individuals. Interestingly, migraine without aura was found as an initial symptom of the disease in two young mutation carriers (22 and 25 years, respectively), who did not show any additional clinical features or any MRI abnormalities. This indicates that migraine without aura in the absence of MRI abnormalities may represent an early initial symptom of CADASIL, which is difficult to diagnose in the absence of molecular diagnosis. Therefore, the used molecular screening method for Notch3 mutations provides a rapid and accurate diagnostic tool in addition to the standard diagnostic procedures.

hKChIP2 is a functional modifier of hKv4.3 potassium channels: cloning and expression of a short hKChIP2 splice variant.

OBJECTIVE: The Ca(2+) independent transient outward K(+) current (I(to1)) in the heart is responsible for the initial phase of repolarization. The hKv4.3 K(+) channel alpha-subunit contributes to the I(to1) current in many regions of the human heart. Consistently, downregulation of hKv4.3 transcripts in heart failure and atrial fibrillation is linked to reduction in I(to1) conductance. The recently cloned KChIP family of calcium sensors has been shown to modulate A-type potassium channels of the Kv4 K(+) channel subfamily. METHODS AND RESULTS: We describe the cloning and tissue distribution of hKChIP2, as well as its functional interaction with hKv4.3 after expression in Xenopus oocytes. Furthermore, we isolated a short splice variant of the hKChIP2 gene (hKCNIP2), which represents the major hKChIP2 transcript. Northern blot analyses revealed that hKChIP2 is expressed in the human heart and occurs in the adult atria and ventricles but not in the fetal heart. Upon coexpression with hKv4.3 both hKChIP2 isoforms increased the current amplitude, slowed the inactivation and increased the recovery from inactivation of hKv4.3 currents. For the first time we analyzed the influence of a KChIP protein on the voltage of half-maximal inactivation of Kv4 channels. We demonstrate that the hKChIP2 isoforms shifted the half-maximal inactivation to more positive potentials, but to a different extent. By elucidating the genomic structure, we provide important information for future analysis of the hKCNIP2 gene in candidate disorders. In the course of this work we mapped the hKCNIP2 gene to chromosome 10q24. CONCLUSIONS: Heteromeric hKv4.3/hKChIP2 currents more closely resemble native epicardial I(to1), suggesting that hKChIP2 is a true beta-subunit of human cardiac I(to1). As a result hKChIP2 might play a role in cardiac diseases, where a contribution of I(to1) has been shown.

Screening of deletions in SMN, NAIP and BTF2p44 genes in Turkish spinal muscular atrophy patients.

Deletions of the spinal muscular atrophy (SMA)-determining gene, SMN1, NAIP, and a third multicopy gene, BTF2p44tel were investigated in 60 unrelated Turkish SMA patients. SMN1 was deleted for at least exons 7 and 8 in 85% of the Turkish SMA patients. The NAIP gene was deleted in 75 and 33% of type I and type II SMA patients, respectively. Analysis of the 5'end of the BTF2p44tel gene indicated the extension of deletion in 13.3% of the cases, mainly in type I patients. Deletions of the NAIP and BTF2p44tel genes were detected in 1.3 and 3.9% of carrriers, respectively, in Turkish SMA families. Two patients were detected to harbor the hybrid SMN gene, one type II with deletion of the NAIP gene, and one type III without deletion of the NAIP gene.

Seven cases of Wiedmann-Beckwith syndrome, including the first reported case of mosaic paternal isodisomy along the whole chromosome 11.

Genomic imprinting of chromosome arm 11p is involved in the Wiedemann-Beckwith syndrome (WBS). About 20% of patients with sporadic WBS have paternal uniparental disomy (UPD) of 11p. Mitotic recombination at the 11p region has been suggested to be responsible for the somatic mosaicism in these patients. Our current study concerning sporadic WBS patients demonstrated six patients with mosaic isodisomy restricted to part of 11p and one patient with mosaic paternal uniparental disomy for the whole chromosome 11. Apparently the clinical findings for this patient did not differ from data reported for other WBS patients. This case makes it unlikely that the proximal short arm and the long arm of chromosome 11 contain imprinted genes with a phenotype recognizable prenatally or in infancy, and gives some support to the hypothesis that non-mosaic UPD-11 is prenatally lethal.

Identification of the parental origin of polysomy in two 49,XXXXY cases.

The parental origin and mechanism of formation of polysomy X were studied in two polysomic cases, using four X-linked restriction fragment length polymorphisms, three (CA)n dinucleotide repeat sequences and one variable number tandem repeat (VNTR) locus as genetic markers. A nonradioactive technique based on the hybridization of the polymerase chain reaction (PCR) product was developed for the analysis of dinucleotide repeats. Segregation analysis using different nonradioactive approaches based on the PCR, revealed that all four X chromosomes were of maternal origin. These data provide additional evidence of an identical mechanism of successive nondisjunctions in maternal meiosis I and II.

Clinical, ultrastructural and biochemical studies in two sibs with Ehlers-Danlos syndrome type VI-B-like features.

Two Turkish sibs with clinical features of Ehlers-Danlos syndrome type VI-B are presented. The hydroxylysine contents of dermis and gel electrophoresis of type I and type III collagen produced by fibroblasts were normal. Ultrastructural studies of skin collagen and elastic fibers showed discrete abnormalities. Other syndromes with similar clinical, biochemical and ultrastructural features are discussed.

Tetra-amelia, lung hypo-/aplasia, cleft lip-palate, and heart defect: a new syndrome?

We report on a family with two sons affected with tetra-amelia, cleft lip-palate, bilateral agenesis of lungs, and heart defects. These two cases support the previous suggestions that this complex entity may indeed represent a new syndrome. However, the mode of inheritance is still not clarified.

A somatic origin of homologous Robertsonian translocations and isochromosomes.

One t(14q14q), three t(15q15q), two t(21q21q), and two t(22q22q) nonmosaic, apparently balanced, de novo Robertsonian translocation cases were investigated with polymorphic markers to establish the origin of the translocated chromosomes. Four cases had results indicative of an isochromosome: one t(14q14q) case with mild mental retardation and maternal uniparental disomy (UPD) for chromosome 14, one t(15q15q) case with the Prader-Willi syndrome and UPD(15), a phenotypically normal carrier of t(22q22q) with maternal UPD(22), and a phenotypically normal t(21q21q) case of paternal UPD(21). All UPD cases showed complete homozygosity throughout the involved chromosome, which is supportive of a postmeiotic origin. In the remaining four cases, maternal and paternal inheritance of the involved chromosome was found, which unambiguously implies a somatic origin. One t(15q15q) female had a child with a ring chromosome 15, which was also of probable postmeiotic origin as recombination between grandparental haplotypes had occurred prior to ring formation. UPD might be expected to result from de novo Robertsonian translocations of meiotic origin; however, all de novo homologous translocation cases, so far reported, with UPD of chromosomes 14, 15, 21, or 22 have been isochromosomes. These data provide the first direct evidence that nonmosaic Robertsonian translocations, as well as isochromosomes, are commonly the result of a mitotic exchange.