New insights into cystinuria: 40 new mutations, genotype-phenotype correlation, and digenic inheritance causing partial phenotype

Objective: To clarify the genotype–phenotype correlation and elucidate the role of digenic inheritance in cystinuria. Methods: 164 probands from the International Cystinuria Consortium were screened for mutations in SLC3A1 (type A) and SLC7A9 (type B) and classified on the basis of urine excretion of cystine and dibasic amino acids by obligate heterozygotes into 37 type I (silent heterozygotes), 46 type non-I (hyperexcretor heterozygotes), 14 mixed, and 67 untyped probands. Results: Mutations were identified in 97% of the probands, representing 282 alleles (86.8%). Forty new mutations were identified: 24 in SLC3A1 and 16 in SLC7A9. Type A heterozygotes showed phenotype I, but mutation DupE5-E9 showed phenotype non-I in some heterozygotes. Type B heterozygotes showed phenotype non-I, with the exception of 10 type B mutations which showed phenotype I in some heterozygotes. Thus most type I probands carried type A mutations and all type non-I probands carried type B mutations. Types B and A mutations contributed to mixed type, BB being the most representative genotype. Two mixed cystinuria families transmitted mutations in both genes: double compound heterozygotes (type AB) had greater aminoaciduria than single heterozygotes in their family. Conclusions: Digenic inheritance is an exception (two of 164 families), with a limited contribution to the aminoaciduria values (partial phenotype) in cystinuria. Further mutational analysis could focus on one of the two genes (SLC3A1 preferentially for type I and SLC7A9 for type non-I probands), while for mixed probands analysis of both genes might be required, with priority given to SLC7A9.     

Functional analysis of a new splice site mutation, c.605-3C>A, in the cystinuria gene SLC7A9 leading to exon skipping

Cystinuria is a hereditary disorder of cystine and dibasic amino acid transport across the luminal membrane of renal tubules and intestine, resulting in recurrent nephrolithiasis. While mutations in the SLC3A1 gene cause type I cystinuria, patients with non-type I cystinuria carry mutations in the SLC7A9 gene. Up to now, more than 80 mutations in SLC3A1 and 50 in SLC7A9 have been reported in the literature. While deletions, duplications, and truncating mutations can often unambiguously classified to be pathogenic, the functional relevance of base pair substitutions is often difficult to predict. To determine the functional relevance of a new splice site mutation in intron 5 of SLC7A9, c.605-3C>A, we transfected COS7 cells with expression constructs containing the wild-type and mutant allele, respectively. cDNAs derived from the resulting SLC7A9 mRNAs were sequenced. By this approach we could demonstrate that the mutant allele c.605-3A causes exon skipping and therefore represents a splice site mutation. To the best of our knowledge, this is the first splice site mutation in a cystinuria gene with a proven functional consequence.     

DHCR7 mutations and genotype-phenotype correlation in 37 Polish patients with Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder of cholesterol biosynthesis caused by mutations in the DHCR7 gene. Thirty-seven ethnic Polish patients with SLOS underwent mutation analysis. The mutation frequencies in Polish patients were significantly different from those observed in Western European populations. Two mutations, W151X (22/68 alleles, 32%) and V326L (19/68 alleles, 28%), accounted for 60% of all observed in our cohort. Two missense mutations L68P and L360P have not been reported previously. In total, we report 15 DHCR7 mutations identified in Polish patients. By comparing clinical severity scores and the biochemical and molecular data, a genotype-phenotype correlation was attempted. In compound heterozygotes with one null mutation, the phenotype severity depends on the localization and type of the second mutation: mild phenotypes are correlated with mutations affecting the putative transmembrane domains TM1-TM6 or CT regions and severe phenotypes with mutations localized in TM7 and 4L region. The phenotypic differences of patients with the same genotype suggest that severity of the disease may be affected by other factors.     

Functional analysis of mutations in the OCTN2 transporter causing primary carnitine deficiency: lack of genotype-phenotype correlation

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation caused by defective carnitine transport. This disease is caused by mutations in the novel organic cation transporter OCTN2 (SLC22A5 gene). The disease can present early in life with hypoketotic hypoglycemia or later in life with skeletal myopathy or cardiomyopathy. To determine whether the variation in phenotypic severity is due to mutations retaining residual function, we extended mutational analysis of OCTN2 to four additional European families with primary carnitine deficiency. Three patients were homozygous for novel missense mutations (R169W, G242V, A301D). The fourth patient was compound heterozygous for R169W and W351R substitutions. Stable expression of all the mutations in CHO cells confirmed that all mutations abolished carnitine transport, with the exception of the A301D mutation in which residual carnitine transport was 2-3% of the value measured in cells expressing the normal OCTN2 cDNA. Analysis of the patients characterized in molecular detail by our laboratory failed to indicate a correlation between residual carnitine transport and severity of the phenotype or age at presentation.     

Identification of 12 novel mutations in the SLC3A1 gene in Swedish cystinuria patients

Cystinuria is an autosomal recessive disorder that affects luminal transport of cystine and dibasic amino acids in the kidneys and the small intestine. Three subtypes of cystinuria can be defined biochemically, and the classical form (type I) has been associated with mutations in the amino acid transporter gene SLC3A1. The mutations detected in SLC3A1 tend to be population specific and have not been previously investigated in Sweden. We have screened the entire coding sequence and the intron/exon boundaries of the SLC3A1 gene in 53 cystinuria patients by means of single strand conformation polymorphism (SSCP) and DNA sequencing. We identified 12 novel mutations (a 2 bp deletion, one splice site mutation, and 10 missense mutations) and detected another three mutations that were previously reported. Five polymorphisms were also identified, four of which were formerly described. The most frequent mutation in this study was the previously reported M467T and it was also detected in the normal population with an allelic frequency of 0.5%. Thirty‐seven patients were homozygous for mutations in the SLC3A1 gene and another seven were heterozygous which implies that other genes may be involved in cystinuria. Future investigation of the non‐type I cystinuria gene SLC7A9 may complement our results but recent studies also suggest the presence of other potential disease genes. Hum Mutat 18:516–525, 2001. © 2001 Wiley‐Liss, Inc.     

Functional analysis of nonsynonymous single nucleotide polymorphisms in human SLC26A9

Slc26 anion transporters play crucial roles in transepithelial Cl(-) absorption and HCO(3)(-) secretion; Slc26 protein mutations lead to several diseases. Slc26a9 functions as a Cl(-) channel and electrogenic Cl(-)--HCO(3)(-) exchanger, and can interact with cystic fibrosis transmembrane conductance regulator. Slc26a9(-/-) mice have reduced gastric acid secretion, yet no human disease is currently associated with SLC26A9 coding mutations. Therefore, we tested the function of nonsynonymous, coding, single nucleotide polymorphisms (cSNPs) of SLC26A9. Presently, eight cSNPs are NCBI documented: Y70N, T127N, I384T, R575W, P606L, V622L, V744M, and H748R. Using two-electrode voltage-clamp and anion selective electrodes, we measured the biophysical consequences of these cSNPs. Y70N (cytoplasmic N-terminus) displays higher channel activity and enhanced Cl(-)--HCO(3)(-) exchange. T127N (transmembrane) results in smaller halide currents but not for SCN(-). V622L (STAS domain) and V744M (STAS adjacent) decreased plasma membrane expression, which partially accounts for decreased whole cell currents. Nevertheless, V622L transport is reduced to ～50%. SLC26A9 polymorphisms lead to several function modifications (increased activity, decreased activity, altered protein expression), which could lead to a spectrum of pathophysiologies. Thus, knowing an individual's SLC26A9 genetics becomes important for understanding disease potentially caused by SLC26A9 mutations or modifying diseases, for example, cystic fibrosis. Our results also provide a framework to understand SLC26A9 transport modalities and structure-function relationships.     

Identification of novel SLC3A1 gene mutations in Spanish cystinuria families and association with clinical phenotypes

Cystinuria is an inherited metabolic disease characterized by an abnormal urinary excretion of cystine and dibasic amino acids, leading to kidney stone formation. Incidence of cystinuria in the Mediterranean Spanish population is one of the highest in the world. In view of the low prevalence of previously reported mutations in the SLC3A1 gene, analyses to identify novel variants were carried out on 20 cystinuria families. Additionally, we investigated the possible association between these molecular variants and clinical phenotypes. Genomic DNA from 48 cystinuria patients, 44 healthy relatives and 81 unrelated controls from the East Mediterranean coast of Spain was screened by conformation sensitive gel electrophoresis. Abnormal patterns were confirmed by nucleotide sequence determination and by further restriction fragment-length polymorphism. We only found 11 genetic variants within the SLC3A1 gene: five known polymorphisms (114C > A, 231T > A, 1136 + 3delT, 1332 + 7T > C and 1338G > A), four point mutations (M467T, R452W, I105R and Y461X), one single base pair deletion (1767delA) and one 2-bp insertion (1670insAT). Two of these genetic variants (I105R and 1670insAT) were described for the first time. All mutations but one were detected in families classified as Type I cystinuria due to the transmission pattern of the disease. Association analyses revealed that 231T > A (M467T), 1136 + 3delT and 1332 + 7T > C genetic variants were statistically related with urinary amino acid excretion in cystinuria patients. Although some molecular variants within the SLC3A1 gene were associated with clinical traits in cystinuria patients, the low detection rate of mutations in this gene strongly suggests that variation of the SLC3A1 is not the major genetic factor contributing to cystinuria in this Mediterranean population.     

Mutational analysis of ATP7B and genotype-phenotype correlation in Japanese with Wilson's disease

The gene ATP7B responsible for Wilson's disease (WD) produces a protein which is predicted to be a copper-binding P-type ATPase, homologous to the Menkes disease gene (ATP7A). Various mutations of ATP7B have been identified. This study aimed to detect disease-causing mutations, to clarify their frequency and distribution, to determine whether genotype correlates with phenotype, and to determine the rate of abnormal findings in heterozygotes for the WD gene. We analyzed 41 unrelated Japanese WD families, including 47 patients. Twenty-one mutations, including nine novel ones, were identified. 2871delC (15.9%), 1708-5T-->G (11. 0%), and Arg778Leu (13.4%) were the most common mutations. 2871delC was detected mainly in eastern Japan and 1708-5T-->G in western Japan. The homozygotes for the 1708-5T-->G, 2871delC, or Arg778Leu mutations did not show a correlation with their phenotypes. Ceruloplasmin and copper levels were abnormally low in 28.6% and 35. 0% of heterozygotes, respectively. When patients and their families are screened for WD, a high rate of abnormal laboratory data in heterozygotes must be taken into account.     

Spectrum of mutations and genotype-phenotype analysis in Currarino syndrome

The triad of a presacral tumour, sacral agenesis and anorectal malformation constitutes the Currarino syndrome which is caused by dorsal-ventral patterning defects during embryonic development. The syndrome occurs in the majority of patients as an autosomal dominant trait associated with mutations in the homeobox gene HLXB9 which encodes the nuclear protein HB9. However, genotype-phenotype analyses have been performed only in a few families and there are no reports about the specific impact of HLXB9 mutations on HB9 function. We performed a mutational analysis in 72 individuals from nine families with Currarino syndrome. We identified a total of five HLXB9 mutations, four novel and one known mutation, in four out of four families and one out of five sporadic cases. Highly variable phenotypes and a low penetrance with half of all carriers being clinically asymptomatic were found in three families, whereas affected members of one family showed almost identical phenotypes. However, an obvious genotype-phenotype correlation was not found. While HLXB9 mutations were diagnosed in 23 patients, no mutation or microdeletion was detected in four sporadic patients with Currarino syndrome. The distribution pattern of here and previously reported HLXB9 mutations indicates mutational predilection sites within exon 1 and the homeobox. Furthermore, sequence homology to Drosophila homeobox genes suggest that some of these mutations located within the homeobox may alter the DNA-binding specificity of HB9 while those in sequences homologous to a recently identified NLS motif of the human homeobox gene PDX-1 may impair nuclear translocation of the mutated protein.     

Pendred syndrome, DFNB4, and PDS/SLC26A4 identification of eight novel mutations and possible genotype-phenotype correlations

Mutations in PDS (SLC26A4) cause both Pendred syndrome and DFNB4, two autosomal recessive disorders that share hearing loss as a common feature. The hearing loss is associated with temporal bone abnormalities, ranging from isolated enlargement of the vestibular aqueduct (dilated vestibular aqueduct, DVA) to Mondini dysplasia, a complex malformation in which the normal cochlear spiral of 2(1/2) turns is replaced by a hypoplastic coil of 1(1/2) turns. In Pendred syndrome, thyromegaly also develops, although affected persons usually remain euthyroid. We identified PDS mutations in the proband of 14 of 47 simplex families (30%) and nine of 11 multiplex families (82%) (P=0.0023). In all cases, mutations segregated with the disease state in multiplex families. Included in the 15 different PDS allele variants we found were eight novel mutations. The two most common mutations, T416P and IVS8+1G>A, were present in 22% and 30% of families, respectively. The finding of PDS mutations in five of six multiplex families with DVA (83%) and four of five multiplex families with Mondini dysplasia (80%) implies that mutations in this gene are the major genetic cause of these temporal anomalies. Comparative analysis of phenotypic and genotypic data supports the hypothesis that the type of temporal bone anomaly may depend on the specific PDS allele variant present.     

Identification of novel mutations in WFS1 and genotype-phenotype correlation in Wolfram syndrome

Mutations in the WFS1 gene have been reported in Wolfram syndrome (WS), an autosomal recessive disorder defined by early onset of diabetes mellitus (DM) and progressive optic atrophy. Because of the low prevalence of this syndrome and the recent identification of the WFS1 gene, few data are available concerning the relationships between clinical and molecular aspects of the disease. Here, we describe 12 patients from 11 families with WS. We report on eight novel (A214fsX285, L293fsX303, P346L, I427S, V503fsX517, R558C, S605fsX711, P838L) and seven previously reported mutations. We also looked for genotype-phenotype correlation both in patients included in this study and 19 additional WS patients that were previously reported. Subsequently, we performed a systematic review and meta-analysis of five published clinical and molecular studies of WFS1 for genotype-phenotype correlation, combined with our current French patient group for a total of 96 patients. The presence of two inactivating mutations was shown to predispose to an earlier age of onset of both DM and optic atrophy. Moreover, the clinical expression of WS was more complete and occurred earlier in patients harboring no missense mutation.     

LRRK2 and Parkin mutations in a family with parkinsonism-Lack of genotype-phenotype correlation

Here we report the relationship between age at onset, clinical course and genotype in a family with combined LRRK2 G2019S and Parkin exon 2 deletions. In the combined mutation carriers the age at onset and clinical course was highly variable and not always younger than in the carriers of LRRK2 G2019S mutations alone.     

Molecular analysis of Japanese patients with Rett syndrome: Identification of five novel mutations and genotype-phenotype correlation

Rett syndrome is an X-linked dominant neurodevelopmental disorder that affects females almost exclusively. The recent identification of mutations of the methyl-CpG-binding protein 2 gene (MECP2) in patients with RTT, encouraged us to analyze the gene in 37 Japanese patients divided into classical RTT (14 cases), variant RTT (13 cases), and mentally retarded patients with Rett-like features (10 cases). Mutations in MECP2 were identified from most of the patients with classical and variant RTT (25 of 27 cases). Six reported common mutations were detected in 17 cases, and rare single nucleotide substitutions were found in 3 patients. In addition, one insertion mutation (1189insA) and four deletion mutations including one double deletion mutant (451delG, 100del4, 1124del53 and 881del289 plus 1187del8) were newly identified. In the 10 mentally retarded patients with Rett-like features, however, no mutation was detected in the coding region of MECP2. The finding of MECP2 mutations in 92.5% of patients with RTT indicates that RTT fulfilling the diagnostic criteria are due to genetic alteration.     

Molecular Genetic Analysis of SLC3A1 and SLC7A9 Genes in Czech and Slovak Cystinuric Patients

Cystinuria is a frequently inherited metabolic disorder in the Czech population (frequency 1/5,600) caused by a defect in the renal transport of cystine and dibasic amino acids (arginine, lysine and ornithine). The disease is characterized by increased urinary excretion of the amino acids and often leads to recurrent nephrolithiasis. Cystinuria is classified into two subtypes (type I and type non‐I). Type I is caused predominantly by mutations in the SLC3A1 gene (2p16.3), encoding heavy subunit (rBAT) of the heterodimeric transporter. Cystinuria non‐I type is caused by mutations in the SLC7A9 gene (19q13.1). In this study, we present results of molecular genetic analysis of the SLC3A1 and the SLC7A9 genes in 24 unrelated cystinuria families. Individual exons of the SLC3A1 and SLC7A9 genes were analyzed by direct sequencing. We found ten different mutations in the SLC3A1 gene including six novel ones: three missense mutations (G140R), D179Y and R365P), one splice site mutation (1137‐2A>G), one deletion (1515_1516delAA), and one nonsense mutation (Q119X). The most frequent mutation, M467T; was detected in 36% of all type I classified alleles. In the SLC7A9 gene we found six mutations including three new ones: one missense mutation (G319R), one insertion (611_612insA) and one deletion (205_206delTG). One patient was compound heterozygote for one SLC3A1 and one SLC7A9 mutation. Our results confirm that cystinuria is a heterogeneous disorder at the molecular level, and contribute to the understanding of the distribution and frequency of mutations causing cystinuria in the Caucasian population.