Mutations in the tight-junction gene claudin 19 (CLDN19) are associated with renal magnesium wasting, renal failure, and severe ocular involvement

Claudins are major components of tight junctions and contribute to the epithelial-barrier function by restricting free diffusion of solutes through the paracellular pathway. We have mapped a new locus for recessive renal magnesium loss on chromosome 1p34.2 and have identified mutations in CLDN19, a member of the claudin multigene family, in patients affected by hypomagnesemia, renal failure, and severe ocular abnormalities. CLDN19 encodes the tight-junction protein claudin-19, and we demonstrate high expression of CLDN19 in renal tubules and the retina. The identified mutations interfere severely with either cell-membrane trafficking or the assembly of the claudin-19 protein. The identification of CLDN19 mutations in patients with chronic renal failure and severe visual impairment supports the fundamental role of claudin-19 for normal renal tubular function and undisturbed organization and development of the retina.     

Genetic Analysis of Genes Related to Tight Junction Function in the Korean Population with Non-Syndromic Hearing Loss

Tight junctions (TJs) are essential components of eukaryotic cells, and serve as paracellular barriers and zippers between adjacent tissues. TJs are critical for normal functioning of the organ of Corti, a part of the inner ear that causes loss of sensorineural hearing when damaged. To investigate the relation between genes involved in TJ function and hereditary loss of sensorineural hearing in the Korean population, we selected the TJP2 and CLDN14 genes as candidates for gene screening of 135 Korean individuals. The TJP2 gene, mutation of which causes autosomal dominant non-syndromic hearing loss (ADNSHL), lies at the DFNA51 locus on chromosome 9. The CLDN14 gene, mutation of which causes autosomal recessive non-syndromic hearing loss (ARNSHL), lies at the DFNB29 locus on chromosome 21. In the present study, we conducted genetic analyses of the TJP2 and CLDN14 genes in 87 unrelated patients with ADNSHL and 48 unrelated patients with either ARNSHL or potentially sporadic hearing loss. We identified two pathogenic variations, c.334G>A (p.A112T) and c.3562A>G (p.T1188A), and ten single nucleotide polymorphisms (SNPs) in the TJP2 gene. We found eight non-pathogenic variations in the CLDN14 gene. These findings indicate that, whereas mutation of the TJP2 gene might cause ADNSHL, CLDN14 is not a major causative gene for ARNSHL in the Korean population studied. Our findings may improve the understanding of the genetic cause of non-syndromic hearing loss in the Korean population.     

Identification of 99 novel mutations in a worldwide cohort of 1,056 patients with a nephronophthisis-related ciliopathy

Nephronophthisis-related ciliopathies (NPHP-RC) are autosomal-recessive cystic kidney diseases. More than 13 genes are implicated in its pathogenesis to date, accounting for only 40 % of all cases. High-throughput mutation screenings of large patient cohorts represent a powerful tool for diagnostics and identification of novel NPHP genes. We here performed a new high-throughput mutation analysis method to study 13 established NPHP genes (NPHP1–NPHP13) in a worldwide cohort of 1,056 patients diagnosed with NPHP-RC. We first applied multiplexed PCR-based amplification using Fluidigm Access-Array? technology followed by barcoding and next-generation resequencing on an Illumina platform. As a result, we established the molecular diagnosis in 127/1,056 independent individuals (12.0 %) and identified a single heterozygous truncating mutation in an additional 31 individuals (2.9 %). Altogether, we detected 159 different mutations in 11 out of 13 different NPHP genes, 99 of which were novel. Phenotypically most remarkable were two patients with truncating mutations in INVS/NPHP2 who did not present as infants and did not exhibit extrarenal manifestations. In addition, we present the first case of Caroli disease due to mutations in WDR19/NPHP13 and the second case ever with a recessive mutation in GLIS2/NPHP7. This study represents the most comprehensive mutation analysis in NPHP-RC patients, identifying the largest number of novel mutations in a single study worldwide.     

Founder mutations in Tunisia: implications for diagnosis in North Africa and Middle East

Cystinuria (OMIM 220100) is an inborn congenital disorder characterised by a defective cystine metabolism resulting in the formation of cystine stones. Among the heterogeneous group of kidney stone diseases, cystinuria is the only disorder which is exclusively caused by gene mutations. So far, two genes responsible for cystinuria have been identified: SLC3A1 (chromosome 2p21) encodes the heavy subunit rBAT of a renal b^0,+ transporter while SLC7A9 (chromosome 19q12) encodes its interacting light subunit b^0,+AT. Mutations in SLC3A1 are generally associated with an autosomal-recessive mode of inheritance whereas SLC7A9 variants result in a broad clinical variability even within the same family. The detection rate for mutations in these genes is larger than 85%, but it is influenced by the ethnic origin of a patient and the pathophysiological significance of the mutations. In addition to isolated cystinuria, patients suffering from the hypotonia-cystinuria syndrome have been reported carrying deletions including at least the SLC3A1 and the PREPL genes in 2p21. By extensive molecular screening studies in large cohort of patients a broad spectrum of mutations could be identified, several of these variants were functionally analysed and thereby allowed insights in the pathology of the disease as well as in the renal trafficking of cystine and the dibasic amino acids. In our review we will summarize the current knowledge on the physiological and the genetic basis of cystinuria as an inborn cause of kidney stones, and the application of this knowledge in genetic testing strategies.     

The First Nationwide Survey and Genetic Analyses of Bardet-Biedl Syndrome in Japan

Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder characterized by central obesity, mental impairment, rod-cone dystrophy, polydactyly, hypogonadism in males, and renal abnormalities. The causative genes have been identified as BBS1-19. In Western countries, this disease is often reported, but remains undiagnosed in many patients until later in life, while only a few patients with no mutations identified have been reported in Japan. We thus conducted the first nationwide survey of BBS in Japan by sending questionnaires to 2,166 clinical departments with board-certified specialists and found 7 patients with clinically definite BBS. We performed exome analyses combined with analyses of mRNA and protein in these patients. We identified 2 novel mutations in the BBS5 gene (p.R89X and IVS7-27 T>G) in 2 sibling patients. The latter mutation that resided far from the authentic splicing site was associated with skipping of exon 8. We also found 3 previously reported mutations in the BBS2 (p.R413X and p.R480X) and BBS7 (p.C243Y) genes in 2 patients. To our knowledge, a nationwide survey of BBS has not been reported in any other country. In addition, this is the first study to identify genetic alterations in Japanese patients with BBS. Our results indicate that BBS in Japan is genetically heterogeneous and at least partly shares genetic features with BBS in other countries.     

IDH1 and IDH2 mutations are frequent in Chinese patients with acute myeloid leukemia but rare in other types of hematological disorders

Frequent mutations in the isocitrate dehydrogenase 1 and 2 genes (IDH1 and IDH2) have been identified in gliomas and acute myeloid leukemia (AML). Our aim is to assess whether IDH mutations were presented in Chinese patients with various hematological disorders. In this study, we screened the IDH1 and IDH2 mutations in a cohort of 456 Chinese patients with various hematological malignancies and disorders. We found three missense (p.R132C, p.R132G, and p.I99M; occurred in five patients) and one silent mutation (c.315C>T; occurred in two patients) in the IDH1 gene and two missense mutations (p.R140Q and p.R172K; occurred in four AML patients) and one silent mutation (c.435G>A) in the IDH2 gene. Except for one non-Hodgkin lymphoma (NHL) patient harboring IDH1 mutation p.R132C, all IDH1 and IDH2 missense mutations were observed in patients with AML. Intriguingly, the IDH2 mutation p.R140Q and novel IDH1 mutation p.I99M co-occurred in a 75-year-old patient with AML developed from myelodysplastic syndromes (MDS). The frequency of IDH1 and IDH2 missense mutations in Chinese AML patients reached 5.9% and 8.3%, respectively. Our results supported the recent findings that IDH gene mutations were common in AML. Conversely, IDH mutations were rather rare in Chinese patients with other types of hematological disorders.     

Mutation Screening of the <Emphasis Type="Italic">CHCHD10</Emphasis> Gene in Chinese Patients with Amyotrophic Lateral Sclerosis

Mutations in the coiled-coil-helix-coiled-coil-helix domain-containing protein 10 gene (CHCHD10), involved in mitochondrial function, have recently been reported as a causative gene of amyotrophic lateral sclerosis (ALS). The aim of this study was to obtain the mutation prevalence of CHCHD10 and the phenotypes with mutations in Chinese ALS patients. A cohort of 499 ALS patients including 487 sporadic ALS (SALS) and 12 familial ALS (FALS), from the Department of Neurology, West China Hospital of Sichuan University, were screened for mutations of all exons of the CHCHD10 gene by Sanger sequencing. Novel candidate mutations or variants were confirmed by polymerase chain reaction-restriction fragment length polymorphism in 466 healthy individuals. All patients identified with mutations of CHCHD10 gene were screened for mutations of the common ALS causative genes including C9orf72, SOD1, TARDBP, FUS, PFN1, and SQSTM1. Three heterozygous variants, including two missense mutations (c.275A?>?G (p.Y92C) and c.306G?>?C (p.Q102H)) and a synonymous change c.306G?>?A (p.Q102Q), were found in exon 3 of CHCHD10 in three alive SALS individuals (with the longest disease duration of 8.6 years), all of which were not detected in healthy controls. No mutation in CHCHD10 was identified in FALS patients. No mutation was found in the aforementioned common ALS causative genes in the patients who carried CHCHD10 mutations. The mutation frequency of CHCHD10 (0.4 %, 2/487) in a Chinese SALS population suggests CHCHD10 gene mutation appears to be an uncommon cause of ALS in Chinese populations. CHCHD10 mutations are associated with a slow progression and long disease duration.     

Polymorphisms in CaSR and CLDN14 Genes Associated with Increased Risk of Kidney Stone Disease in Patients from the Eastern Part of India

Kidney stone disease (KSD) is a major clinical problem imposing a large burden for both healthcare and economy globally. In India, the prevalence of kidney stone disease is rapidly increasing. This study aimed to evaluate the association between genetic defects in vitamin D receptor (VDR), calcium sensing receptor (CaSR) and claudin 14 (CLDN14) genes and kidney stone disease in patients from eastern India. We enrolled 200 consecutive kidney stone patients (age 18–60 years) (cases) and their corresponding sex and age matched 200 normal individuals (controls). To identify genetic variants responsible for KSD, we performed sequence analysis of VDR, CaSR and CLDN14 genes. Four non-synonymous (rs1801725, rs1042636, rs1801726 and rs2228570), one synonymous (rs219780) and three intronic single nucleotide polymorphisms (SNPs) (rs731236, rs219777 and rs219778) were identified. Genotype and allele frequency analysis of these SNPs revealed that, rs1801725 (Ala986Ser), rs1042636 (Arg990Gly) of CaSR gene and rs219778, rs219780 (Thr229Thr) of CLDN14 gene were significantly associated with KSD. Serum calcium levels were significantly higher in subjects carrying 986Ser allele and calcium excretion was higher in subjects bearing 990Gly allele. In conclusion, rs1801725, rs1042636, rs219778 and rs219780 SNPs were associated with kidney stone risk in patients from the eastern part of India.     

Tight Junctional Localization of Claudin-16 Is Regulated by Syntaxin 8 in Renal Tubular Epithelial Cells

Claudin-16 (CLDN16) regulates the paracellular reabsorption of Mg²⁺ in the thick ascending limb of Henle''s loop. However, the mechanism regulating the tight junctional localization of CLDN16 remains unknown. In yeast two-hybrid systems, we found that CLDN16 bound to syntaxin 8 (STX8), a target soluble N-ethylmaleimide-sensitive factor attachment protein receptor. We have examined the effect of STX8 on the localization and function of CLDN16 using Madin-Darby canine kidney cells expressing FLAG-tagged CLDN16. A pulldown assay showed that the carboxyl cytoplasmic region of human CLDN16 bound to STX8. CLDN16 was localized in the thick ascending limb, whereas STX8 was widely distributed throughout the rat kidney. An association between CLDN16 and STX8 was observed in rat renal homogenates and Madin-Darby canine kidney cells. STX8 siRNA decreased the cell surface localization of CLDN16 and transepithelial electrical resistance and permeability to Mg²⁺ but increased the co-localization of CLDN16 with early endosome and lysosome markers. Dephosphorylation of CLDN16 by protein kinase A inhibitors and S217A mutant, a dephosphorylated form, decreased the association with STX8 and the cell surface localization of CLDN16. Recycling assays indicated that STX8 siRNA decreased the trafficking of CLDN16 to the plasma membrane without affecting endocytosis. Dominant negative Rab11 and recycling inhibitor primaquine decreased the cell surface localization of CLDN16, which was similar to that in STX8 siRNA-transfected cells. These results suggest that STX8 mediates the recycling of CLDN16 and constitutes an important component of the CLDN16 trafficking machinery in the kidney.     

Claudin-7 is frequently overexpressed in ovarian cancer and promotes invasion

Background

Claudins are tight junction proteins that are involved in tight junction formation and function. Previous studies have shown that claudin-7 is frequently upregulated in epithelial ovarian cancer (EOC) along with claudin-3 and claudin-4. Here, we investigate in detail the expression patterns of claudin-7, as well as its possible functions in EOC.

Methodology/Principal Findings

A total of 95 ovarian tissue samples (7 normal ovarian tissues, 65 serous carcinomas, 11 clear cell carcinomas, 8 endometrioid carcinomas and 4 mucinous carcinomas) were studied for claudin-7 expression. In real-time RT-PCR analysis, the gene for claudin-7, CLDN7, was found to be upregulated in all the tumor tissue samples studied. Similarly, immunohistochemical analysis and western blotting showed that claudin-7 protein was significantly overexpressed in the vast majority of EOCs. Small interfering RNA-mediated knockdown of claudin-7 in ovarian cancer cells led to significant changes in gene expression as measured by microarrays and validated by RT-PCR and immunoblotting. Analyses of the genes differentially expressed revealed that the genes altered in response to claudin-7 knockdown were associated with pathways implicated in various molecular and cellular functions such as cell cycle, cellular growth and proliferation, cell death, development, and cell movement. Through functional experiments in vitro, we found that both migration and invasion were altered in cells where CLDN7 had been knocked down or overexpressed. Interestingly, claudin-7 expression was associated with a net increase in invasion, but also with a decrease in migration.

Conclusion/Significance

Our work shows that claudin-7 is significantly upregulated in EOC and that it may be functionally involved in ovarian carcinoma invasion. CLDN7 may therefore represent potential marker for ovarian cancer detection and a target for therapy.     

Frequency of COL4A3/COL4A4 Mutations amongst Families Segregating Glomerular Microscopic Hematuria and Evidence for Activation of the Unfolded Protein Response. Focal and Segmental Glomerulosclerosis Is a Frequent Development during Ageing

Familial glomerular hematuria(s) comprise a genetically heterogeneous group of conditions which include Alport Syndrome (AS) and thin basement membrane nephropathy (TBMN). Here we investigated 57 Greek-Cypriot families presenting glomerular microscopic hematuria (GMH), with or without proteinuria or chronic kidney function decline, but excluded classical AS. We specifically searched the COL4A3/A4 genes and identified 8 heterozygous mutations in 16 families (28,1%). Eight non-related families featured the founder mutation COL4A3-p.(G1334E). Renal biopsies from 8 patients showed TBMN and focal segmental glomerulosclerosis (FSGS). Ten patients (11.5%) reached end-stage kidney disease (ESKD) at ages ranging from 37-69-yo (mean 50,1-yo). Next generation sequencing of the patients who progressed to ESKD failed to reveal a second mutation in any of the COL4A3/A4/A5 genes, supporting that true heterozygosity for COL4A3/A4 mutations predisposes to CRF/ESKD. Although this could be viewed as a milder and late-onset form of autosomal dominant AS, we had no evidence of ultrastructural features or extrarenal manifestations that would justify this diagnosis. Functional studies in cultured podocytes transfected with wild type or mutant COL4A3 chains showed retention of mutant collagens and differential activation of the unfolded protein response (UPR) cascade. This signifies the potential role of the UPR cascade in modulating the final phenotype in patients with collagen IV nephropathies.     

Epistatic Role of the MYH9/APOL1 Region on Familial Hematuria Genes

Familial hematuria (FH) is explained by at least four different genes (see below). About 50% of patients develop late proteinuria and chronic kidney disease (CKD). We hypothesized that MYH9/APOL1, two closely linked genes associated with CKD, may be associated with adverse progression in FH. Our study included 102 thin basement membrane nephropathy (TBMN) patients with three known COL4A3/COL4A4 mutations (cohort A), 83 CFHR5/C3 glomerulopathy patients (cohort B) with a single CFHR5 mutation and 15 Alport syndrome patients (cohort C) with two known COL4A5 mild mutations, who were categorized as “Mild” (controls) or “Severe” (cases), based on renal manifestations. E1 and S1 MYH9 haplotypes and variant rs11089788 were analyzed for association with disease phenotype. Evidence for association with “Severe” progression in CFHR5 nephropathy was found with MYH9 variant rs11089788 and was confirmed in an independent FH cohort, D (cumulative p value = 0.001, odds ratio = 3.06, recessive model). No association was found with APOL1 gene. Quantitative Real time PCR did not reveal any functional significance for the rs11089788 risk allele. Our results derive additional evidence supporting previous reports according to which MYH9 is an important gene per se, predisposing to CKD, suggesting its usefulness as a prognostic marker for young hematuric patients.     

Targeted Exome Sequencing Integrated with Clinicopathological Information Reveals Novel and Rare Mutations in Atypical,Suspected and Unknown Cases of Alport Syndrome or Proteinuria

We applied customized targeted next-generation exome sequencing (NGS) to determine if mutations in genes associated with renal malformations, Alport syndrome (AS) or nephrotic syndrome are a potential cause of renal abnormalities in patients with equivocal or atypical presentation. We first sequenced 4,041 exons representing 292 kidney disease genes in a Caucasian woman with a history of congenital vesicoureteral reflux (VUR), recurrent urinary tract infections and hydronephrosis who presented with nephrotic range proteinuria at the age of 45. Her biopsy was remarkable for focal segmental glomerulosclerosis (FSGS), a potential complication of longstanding VUR. She had no family history of renal disease. Her proteinuria improved initially, however, several years later she presented with worsening proteinuria and microhematuria. NGS analysis revealed two deleterious COL4A3 mutations, one novel and the other previously reported in AS, and a novel deleterious SALL2 mutation, a gene linked to renal malformations. Pedigree analysis confirmed that COL4A3 mutations were nonallelic and compound heterozygous. The genomic results in conjunction with subsequent abnormal electron microscopy, Collagen IV minor chain immunohistochemistry and progressive sensorineural hearing loss confirmed AS. We then modified our NGS approach to enable more efficient discovery of variants associated with AS or a subset of FSGS by multiplexing targeted exome sequencing of 19 genes associated with AS or FSGS in 14 patients. Using this approach, we found novel or known COL4A3 or COL4A5 mutations in a subset of patients with clinically diagnosed or suspected AS, APOL1 variants associated with FSGS in African Americans and novel mutations in genes associated with nephrotic syndrome. These studies demonstrate the successful application of targeted capture-based exome sequencing to simultaneously evaluate genetic variations in many genes in patients with complex renal phenotypes and provide insights into etiology of conditions with equivocal clinical and pathologic presentations.     

Traditional and targeted exome sequencing reveals common, rare and novel functional deleterious variants in RET-signaling complex in a cohort of living US patients with urinary tract malformations

Signaling by the glial cell line-derived neurotrophic factor (GDNF)-RET receptor tyrosine kinase and SPRY1, a RET repressor, is essential for early urinary tract development. Individual or a combination of GDNF, RET and SPRY1 mutant alleles in mice cause renal malformations reminiscent of congenital anomalies of the kidney or urinary tract (CAKUT) in humans and distinct from renal agenesis phenotype in complete GDNF or RET-null mice. We sequenced GDNF, SPRY1 and RET in 122 unrelated living CAKUT patients to discover deleterious mutations that cause CAKUT. Novel or rare deleterious mutations in GDNF or RET were found in six unrelated patients. A family with duplicated collecting system had a novel mutation, RET-R831Q, which showed markedly decreased GDNF-dependent MAPK activity. Two patients with RET-G691S polymorphism harbored additional rare non-synonymous variants GDNF-R93W and RET-R982C. The patient with double RET-G691S/R982C genotype had multiple defects including renal dysplasia, megaureters and cryptorchidism. Presence of both mutations was necessary to affect RET activity. Targeted whole-exome and next-generation sequencing revealed a novel deleterious mutation G443D in GFRα1, the co-receptor for RET, in this patient. Pedigree analysis indicated that the GFRα1 mutation was inherited from the unaffected mother and the RET mutations from the unaffected father. Our studies indicate that 5?% of living CAKUT patients harbor deleterious rare variants or novel mutations in GDNF-GFRα1-RET pathway. We provide evidence for the coexistence of deleterious rare and common variants in genes in the same pathway as a cause of CAKUT and discovered novel phenotypes associated with the RET pathway.     

Genetic analysis of auditory neuropathy spectrum disorder in the Korean population

Auditory neuropathy spectrum disorder (ANSD) is caused by dys-synchronous auditory neural response as a result of impairment of the functions of the auditory nerve or inner hair cells, or synapses between inner hair cells and the auditory nerve. To identify a causative gene causing ANSD in the Korean population, we conducted gene screening of the OTOF, DIAPH3, and PJVK genes in 19 unrelated Korean patients with ANSD. A novel nonsense mutation (p.Y1064X) and a known pathogenic mutation (p.R1939Q) of the OTOF gene were identified in a patient as compound heterozygote. Pedigree analysis for these mutations showed co-segregation of mutation genotype and the disease in the family, and it supported that the p.Y1064X might be a novel genetic cause of autosomal recessive ANSD. A novel missense variant p.K1017R (c.3050A>G) in the DIAPH3 gene was also identified in the heterozygous state. In contrast, no mutation was detected in the PJVK gene. These results indicate that no major causative gene has been reported to date in the Korean population and that pathogenic mutations in undiscovered candidate genes may have an effect on ANSD.     

Identification of SCN1A and PCDH19 Mutations in Chinese Children with Dravet Syndrome

Background

Dravet syndrome is a severe form of epilepsy. Majority of patients have a mutation in SCN1A gene, which encodes a voltage-gated sodium channel. A recent study has demonstrated that 16% of SCN1A-negative patients have a mutation in PCDH19, the gene encoding protocadherin-19. Mutations in other genes account for only a very small proportion of families. TSPYL4 is a novel candidate gene within the locus 6q16.3-q22.31 identified by linkage study.

Objective

The present study examined the mutations in epileptic Chinese children with emphasis on Dravet syndrome.

Methods

A hundred children with severe epilepsy were divided into Dravet syndrome and non-Dravet syndrome groups and screened for SCN1A mutations by direct sequencing. SCN1A-negative Dravet syndrome patients and patients with phenotypes resembling Dravet syndrome were checked for PCDH19 and TSPYL4 mutations.

Results

Eighteen patients (9 males, 9 females) were diagnosed to have Dravet syndrome. Among them, 83% (15/18) had SCN1A mutations including truncating (7), splice site (2) and missense mutations (6). The truncating/splice site mutations were associated with moderate to severe degree of intellectual disability (p<0.05). During the progression of disease, 73% (11/15) had features fitting into the diagnostic criteria of autism spectrum disorder and 53% (8/15) had history of vaccination-induced seizures. A novel PCDH19 p.D377N mutation was identified in one SCN1A-negative female patient with Dravet syndrome and a known PCDH19 p.N340S mutation in a female non-Dravet syndrome patient. The former also inherited a TSPYL4 p.G60R variant.

Conclusion

A high percentage of SCN1A mutations was identified in our Chinese cohort of Dravet syndrome patients but none in the rest of patients. We demonstrated that truncating/splice site mutations were linked to moderate to severe intellectual disability in these patients. A de novo PCDH19 missense mutation together with an inherited TSPYL4 missense variant were identified in a patient with Dravet syndrome.     

Molecular characterization of Polish patients with familial hypercholesterolemia: novel and recurrentLDLR mutations

Autosomal dominant hypercholesterolemia (ADH) is caused by mutations in the genes coding for the low-density lipoprotein receptor (LDLR), apolipoprotein B-100 (APOB), or proprotein convertase subtilisin/kexin type 9 (PCSK9). In this study, a molecular analysis ofLDLR andAPOB was performed in a group of 378 unrelated ADH patients, to explore the mutation spectrum that causes hypercholesterolemia in Poland. All patients were clinically diagnosed with ADH according to a uniform protocol and internationally accepted WHO criteria. Mutational analysis included all exons, exon-intron boundaries and the promoter sequence of theLDLR, and a fragment of exon 26 ofAPOB. Additionally, the MLPA technique was applied to detect rearrangements withinLDLR. In total, 100 sequence variations were identified in 234 (62%) patients. WithinLDLR, 40 novel and 59 previously described sequence variations were detected. Of the 99LDLR sequence variations, 71 may be pathogenic mutations. The most frequentLDLR alteration was a point mutation p.G592E detected in 38 (10%) patients, followed by duplication of exons 4–8 found in 16 individuals (4.2%). Twenty-five cases (6.6%) demonstrated the p.R3527Q mutation ofAPOB. Our findings imply that major rearrangements of theLDLR gene as well as 2 point mutations (p.G592E inLDLR and p.R3527Q inAPOB) are frequent causes of ADH in Poland. However, the heterogeneity ofLDLR mutations detected in the studied group confirms the requirement for complex molecular studies of Polish ADH patients.     

Kallmann syndrome: mutations in the genes encoding prokineticin-2 and prokineticin receptor-2

Kallmann syndrome combines anosmia, related to defective olfactory bulb morphogenesis, and hypogonadism due to gonadotropin-releasing hormone deficiency. Loss-of-function mutations in KAL1 and FGFR1 underlie the X chromosome-linked form and an autosomal dominant form of the disease, respectively. Mutations in these genes, however, only account for approximately 20% of all Kallmann syndrome cases. In a cohort of 192 patients we took a candidate gene strategy and identified ten and four different point mutations in the genes encoding the G protein-coupled prokineticin receptor-2 (PROKR2) and one of its ligands, prokineticin-2 (PROK2), respectively. The mutations in PROK2 were detected in the heterozygous state, whereas PROKR2 mutations were found in the heterozygous, homozygous, or compound heterozygous state. In addition, one of the patients heterozygous for a PROKR2 mutation was also carrying a missense mutation in KAL1, thus indicating a possible digenic inheritance of the disease in this individual. These findings reveal that insufficient prokineticin-signaling through PROKR2 leads to abnormal development of the olfactory system and reproductive axis in man. They also shed new light on the complex genetic transmission of Kallmann syndrome.     

Multiple Causes for Delay in Arrival at Hospital in Acute Stroke Patients in Aydin, Turkey

Background

Mutations in the genes PRKN and LRRK2 are the most frequent known genetic lesions among Parkinson's disease patients. We have previously reported that in the Portuguese population the LRRK2 c.6055G > A; p.G2019S mutation has one of the highest frequencies in Europe.

Methods

Here, we follow up on those results, screening not only LRRK2, but also PRKN, SNCA and PINK1 in a cohort of early-onset and late-onset familial Portuguese Parkinson disease patients. This series comprises 66 patients selected from a consecutive series of 132 patients. This selection was made in order to include only early onset patients (age at onset below 50 years) or late-onset patients with a positive family history (at least one affected relative). All genes were sequenced bi-directionally, and, additionally, SNCA, PRKN and PINK1 were subjected to gene dosage analysis.

Results

We found mutations both in LRRK2 and PRKN, while the remaining genes yielded no mutations. Seven of the studied patients showed pathogenic mutations, in homozygosity or compound heterozygosity for PRKN, and heterozygosity for LRRK2.

Conclusion

Mutations are common in Portuguese patients with Parkinson's disease, and these results clearly have implications not only for the genetic diagnosis, but also for the genetic counseling of these patients.