Identification of two novel CAKUT-causing genes by massively parallel exon resequencing of candidate genes in patients with unilateral renal agenesis.

Congenital abnormalities of the kidney and urinary tract (CAKUT) are the most frequent cause of chronic kidney disease in children, accounting for about half of all cases. Although many forms of CAKUT are likely caused by single-gene defects, mutations in only a few genes have been identified. In order to detect new contributing genes we pooled DNA from 20 individuals to amplify all 313 exons of 30 CAKUT candidate genes by PCR analysis and massively parallel exon resequencing. Mutation carriers were identified by Sanger sequencing. We repeated the analysis with 20 new patients to give a total of 29 with unilateral renal agenesis and 11 with other CAKUT phenotypes. Five heterozygous missense mutations were detected in 2 candidate genes (4 mutations in FRAS1 and 1 in FREM2) not previously implicated in non-syndromic CAKUT in humans. All of these mutations were absent from 96 healthy control individuals and had a PolyPhen score over 1.4, predicting possible damaging effects of the mutation on protein function. Recessive truncating mutations in FRAS1 and FREM2 were known to cause Fraser syndrome in humans and mice; however, a phenotype in heterozygous carriers has not been described. Thus, heterozygous missense mutations in FRAS1 and FREM2 cause non-syndromic CAKUT in humans.

Mapping of balanced chromosome translocation breakpoints to the basepair level from microdissected chromosomes.

The analysis of structural variants associated with specific phenotypic features is promising for the elucidation of the function of involved genes. There is, however, at present no approach allowing the rapid mapping of chromosomal translocation breakpoints to the basepair level from a single chromosome. Here we demonstrate that we have advanced both the microdissection and the subsequent unbiased amplification to an extent that breakpoint mapping to the basepair level has become possible. As a case in point we analysed the two breakpoints of a t(7;13) translocation observed in a patient with split hand/foot malformation (SHFM1). The amplification products of the der(7) and of the der(13) were hybridized to custom-made arrays, enabling us to define primers at flanking breakpoint regions and thus to fine-map the breakpoints to the basepair level. Consequently, our results will also contribute to a further delineation of causative mechanisms underlying SHFM1 which are currently unknown.

Unravelling the parental origin and mechanism of formation of the 47,XY,i(X)(q10) Klinefelter karyotype variant.

OBJECTIVE: To describe the parental origin and the mechanism of formation of an 47,XY,idic(X)(p11.2) karyotype in a patient with Klinefelter syndrome. DESIGN: Case report. SETTING: A university hospital. PATIENT(S): A 36-year-old man with primary infertility. INTERVENTION(S): Genotype-phenotype correlation and microsatellite marker-mediated haplotype analysis subsequent to whole genome amplification of microdissected chromosomes. MAIN OUTCOME MEASURE(S): Genotype-phenotype correlation, mechanism of formation, and parental origin. RESULT(S): Maternal origin of the isochromosome and the normal X chromosome and loss of maternal heterozygosity for all informative Xq markers on the isochromosome and in each case, the presence of the other maternal allele on the normal homologue was shown. Comparative analysis of the clinical features of 17 additional cases and of 1 case with a 46,XY/47,XY,i(X)(q10) karyotype reported in the literature revealed a phenotype very similar to the clinical findings in patients with a 47,XXY karyotype. CONCLUSION(S): The molecular results in our patient indicate a maternal origin of a true dicentric isochromosome and most likely postzygotic formation subsequent to a nondisjunction in maternal meiosis II. With the exception of the final height the phenotype of Klinefelter syndrome appears not to be the consequence of trisomy of the pseudoautosomal region on Xp.

Yellow teeth, seizures, and mental retardation: a less severe case of Kohlschütter-Tönz syndrome.

We describe an 11-year-old boy with hypoplastic amelogenesis imperfecta, yellow teeth, seizures, and developmental delay, which are the hallmarks of Kohlschütter-Tönz syndrome. Compared to other reported cases of the syndrome, our patient had less severe developmental delay. Also, spasticity and loss of mental capacity should not be considered obligatory manifestations of the syndrome because they are not present in half of reported patients, as well as in our family. Origin of the parents of our patient from neighboring villages supports autosomal recessive inheritance of Kohlschütter-Tönz syndrome.

GJB2 mutations in keratitis-ichthyosis-deafness syndrome including its fatal form.

Keratitis-ichthyosis-deafness syndrome (KID; MIM 148210) is a rare congenital disorder characterized by vascularizing keratitis, sensorineural hearing loss (HL), and progressive erythrokeratoderma. Clinical variability including a fatal course of KID in the first year of life has been reported. Germline missense mutations in GJB2, encoding connexin-26, were recently found to cause KID in 14 unrelated juvenile and adult patients. We identified a de novo GJB2 mutation G45E in a patient displaying the fatal form of the disease. No mutations were detected in five other connexin and mitochondrial genes. The G45E mutation was not reported previously in Caucasian patients but was the third most common GJB2 mutation (16% of disease alleles) in Japanese patients with autosomal recessive non-syndromic HL. This finding suggests different modes of action of the same GJB2 mutation depending on the genetic background. This hypothesis was further substantiated by our observation of a variable clinical course in unrelated KID patients from Austria harboring the common D50N mutation in GJB2.

The 342-kb deletion in GJB6 is not present in patients with non-syndromic hearing loss from Austria.

Recently, a 342-kb deletion involving GJB6 was associated with autosomal-recessive non-syndromic hearing loss (NSHL) and in combination with a GJB2 mutation with digenic NSHL. This deletion was the second most common mutation causing prelingual NSHL in Spain, and was frequently observed in patients from France and Israel. We screened 393 patients with NSHL being negative or heterozygous for GJB2 mutations for this GJB6 deletion using a multiplex PCR. Most patients were of Austrian (84.2%), and the other patients were of Turkish, Serbian, and Bosnian origin. None of these patients was carrying the deletion in GJB6 indicating that the occurrence of this deletion is restricted to certain populations.

Progressive hearing loss, and recurrent sudden sensorineural hearing loss associated with GJB2 mutations--phenotypic spectrum and frequencies of GJB2 mutations in Austria.

Mutations of GJB2 (encoding connexin 26) are the most common cause of hearing loss (HL) in different populations, and a broad spectrum of GJB2 mutations has been identified. We screened 204 consecutive patients with non-syndromic sensorineural hearing loss for GJB2 mutations. Causative GJB2mutations were identified in 31 (15.2%) patients, and two common mutations, c.35delG and L90P (c.269T>C), accounted for 72.1% and 9.8% of GJB2 disease alleles. In four additional patients (2.0%) only one recessive GJB2 mutation was identified, making genetic counselling difficult. No genotype-phenotype correlation was established. We found, however, that homozygotes for truncating mutations were more likely to have a more severe degree of HL compared with other genotypes. Moreover, we showed by co-segregation studies that L90P is a GJB2 disease allele, and that compound heterozygotes for L90P and any recessive mutation share a mild to moderate phenotype. GJB2-associated HL was linked with progressive HL or with recurrent sudden sensorineural hearing loss (SSNHL) in three of 15 cases being analysed retrospectively. We extended the phenotypic spectrum of GJB2-related disease and recommend GJB2 mutation screening also in cases of progressive HL, and recurrent SSNHL. In addition, a carrier frequency of 1/110 (0.9%) for the most common Caucasian mutation in this gene, c.35delG, was determined in 1,212 blood donors from West-Austria, supporting the prevailing hypothesis of a Mediterranean founder mutation. Based on population and patient data, an overall GJB2 mutation carrier frequency of 1.3% was estimated for West-Austria.