Molecular characterization of a large group of Mucopolysaccharidosis type IIIC patients reveals the evolutionary history of the disease

Mucopolysaccharidosis type IIIC (MPSIIIC) is a severe, rare autosomal recessive disorder caused by variants in the heparan‐α‐glucosaminide N‐acetyltransferase (HGSNAT) gene which result in lysosomal accumulation of heparan sulfate. We analyzed clinical presentation, molecular defects and their haplotype context in 78 (27 novel) MPSIIIC cases from 22 countries, the largest group studied so far. We describe for the first time disease‐causing variants in the patients from Brazil, Algeria, Azerbaijan, and Iran, and extend their spectrum within Canada, Colombia, Turkey, and the USA. Six variants are novel: two missense, c.773A>T/p.N258I and c.1267G>T/p.G423W, a nonsense c.164T>A/p.L55*, a splice‐site mutation c.494?1G>A/p.[P165_L187delinsQSCYVTQAGVRWHHLGSLQALPPGFTPFSYLSLLSSWNC,P165fs], a deletion c.1348delG/p.(D450fs) and an insertion c.1479dupA/p.(Leu494fs). The missense HGSNAT variants lacked lysosomal targeting, enzymatic activity, and likely the correct folding. The haplotype analysis identified founder mutations, p.N258I, c.525dupT, and p.L55* in the Brazilian state of Paraiba, c.493+1G>A in Eastern Canada/Quebec, p.A489E in the USA, p.R384* in Poland, p.R344C and p.S518F in the Netherlands and suggested that variants c.525dupT, c.372?2G>A, and c.234+1G>A present in cis with c.564‐98T>C and c.710C>A rare single‐nucleotide polymorphisms, have been introduced by Portuguese settlers in Brazil. Altogether, our results provide insights into the origin, migration roots and founder effects of HGSNAT disease‐causing variants, and reveal the evolutionary history of MPSIIIC.     

Primary hyperoxaluria: Comprehensive mutation screening of the disease causing genes and spectrum of disease-associated pathogenic variants

The primary hyperoxalurias are rare disorders of glyoxylate metabolism. Accurate diagnosis is essential for therapeutic and management strategies. We conducted a molecular study on patients suffering from recurrent calcium-oxalate stones and nephrocalcinosis and screened primary hyperoxaluria causing genes in a large cohort of early-onset cases. Disease-associated pathogenic-variants were defined as missense, nonsense, frameshift-indels, and splice-site variants with a reported minor allele frequency <1% in controls. We found pathogenic-variants in 34% of the cases. Variants in the AGXT gene causing PH-I were identified in 81% of the mutation positive cases. PH-II-associated variants in the GRHPR gene are found in 15% of the pediatric PH-positive population. Only 3% of the PH-positive cases have pathogenic-variants in the HOGA1 gene, responsible to cause PH-III. A population-specific AGXT gene variant c.1049G>A; p.Gly350Asp accounts for 22% of the PH-I-positive patients. Pathogenicity of the identified variants was evaluated by in-silico tools and ACMG guidelines. We have devised a rapid and low-cost approach for the screening of PH by using targeted-NGS highlighting the importance of an accurate and cost-effective screening platform. This is the largest study in Pakistani pediatric patients from South-Asian region that also expands the mutation spectrum of the three known genes.     

Prevalence of MYH-associated polyposis related to three recurrent mutations in Morocco

Background: MYH-associated polyposis (MAP) is an autosomal recessive inherited disease. People with MAP tend to develop multiple adenomatous colon polyps during their lifetime and have an increased risk of colorectal cancer. MAP has only recently been described and there is much to be learned about the condition. Recessively inherited mutations in the base excision repair gene MYH have recently been associated with predisposition to colorectal adenomas and cancer. The epidemiology of MYH-associated polyposis (MAP) is poorly known in populations with high levels of consanguinity like North African populations, in particular in Morocco, and the MAP carrier frequency in the general Moroccan population has never been evaluated. The present study was carried out among the Moroccan population, using molecular epidemiology methods, to estimate the prevalence of homozygote or compound heterozygote genotype conferring MAP due to three mutations reported as recurrent in MAP: c.494A>G (Y165C), c.1145G>A (G382D) and c.1186_1187insGG (p.Glu396fsX42).

Methods: To estimate the prevalence of MYH mutations in Morocco, DNA extracted from blood samples of 400 healthy Moroccans was tested for recurrent MYH mutations using real-time PCR or DNA fragment analysis. Heterozygotes profiles were confirmed by direct sequencing. We searched for the mutations c.494A>G and c.1145G>A in 400 subjects, and the mutation c.1186_1187insGG in 250 subjects.

Results: One subject was heterozygous for c.494A>G (1/400 or 0.25%), three others for c.1145G>A (3/400 or 0.75%) and one was heterozygous for p.Glu396fsX42 (1/250 or 0.4%). The carrier frequency of one of these three mutations in the Moroccan population was calculated to be 1.4% and the frequency of homozygous or compound heterozygote for these three recurrent mutations is 1/10 000.These figures allowed one to estimate at 3500 the number of Moroccans with high risk of developing colon cancer due only to these three recurrent mutations.

Conclusion: This preliminary study shows that the Moroccan population is at risk for MAP. This could help to define diagnosis strategies and patient care and may also have implications for genetic counselling.     

Mutation and haplotype analyses of the <Emphasis Type="Italic">MUT</Emphasis> gene in Japanese patients with methylmalonic acidemia

Methylmalonic acidemia (MMA) is caused by a deficiency in the activity of l-methylmalonyl-CoA mutase (MCM), a vitamin B12 (or cobalamin, Cbl)-dependent enzyme. Apoenzyme-deficient MMA (mut MMA) results from mutations in the nuclear gene MUT. Most of the MUT mutations are thought to be private or restricted to only a few pedigrees. Our group elucidated the spectrum of mutations of Japanese mut MMA patients by performing mutation and haplotype analyses in 29 patients with mut MMA. A sequence analysis identified mutations in 95% (55/58) of the disease alleles. Five mutations were relatively frequent (p.E117X, c.385 + 5G > A, p.R369H, p.L494X, and p.R727X) and four were novel (p.M1V, c.753_753 + 5delGGTATA, c.1560G > C, and c.2098_2099delAT). Haplotype analysis suggested that all of the frequent mutations, with the exception of p.R369H, were spread by the founder effect. Among 46 Japanese patients investigated in the present and previous studies, 76% (70/92) of the mutations were located in exons 2, 6, 8, and 13. This finding – that a limited number of mutations account for most of the mutations in Japanese mut MMA patients – is in contrast with results of a previous study in Caucasian patients.     

Molecular characterization of 22 novel UDP-N-acetylglucosamine-1-phosphate transferase α- and β-subunit (GNPTAB) gene mutations causing mucolipidosis types IIα/β and IIIα/β in 46 patients

Mutational analysis of the GNPTAB gene was performed in 46 apparently unrelated patients with mucolipidosis IIα/β or IIIα/β, characterized by the mistargeting of multiple lysosomal enzymes as a consequence of a UDP-GlcNAc-1-phosphotransferase defect. The GNPTAB mutational spectrum comprised 25 distinct mutant alleles, 22 of which were novel, including 3 nonsense mutations (p.Q314X, p.R375X, p.Q507X), 5 missense mutations (p.I403T, p.C442Y, p.C461G, p.Q926P, p.L1001P), 6 microduplications (c.749dupA, c.857dupA, c.1191_1194dupGCTG, c.1206dupT, c.1331dupG, c.2220_2221dupGA) and 8 microdeletions (c.755_759delCCTCT, c.1399delG, c.1959_1962delTAGT, c.1965delC, c.2550_2554delGAAAA, c.3443_3446delTTTG, c.3487_3490delACAG, c.3523_3529delATGTTCC). All micro-duplications/deletions were predicted to result in the premature termination of translation. A novel exonic SNP (c.303G>A; E101E) was identified which is predicted to create an SFRS1 (SF2/ASF) binding site that may be of potential functional/clinical relevance. This study of mutations in the GNPTAB gene, the largest yet reported, extends our knowledge of the mutational heterogeneity evident in MLIIα/β/MLIIIα/β. © 2009 Wiley-Liss, Inc.     

Molecular analysis and characterization of nine novel CTSK mutations in twelve patients affected by pycnodysostosis

Molecular characterization of twelve unrelated patients affected by the autosomal recessive osteosclerotic skeletal dysplasia, Pycnodysostosis (cathepsin k deficiency), revealed 11 different genotypes. The mutational profile consisted of 12 different mutations, including nine previously unreported ones, spread throughout the whole gene. One mutation occurred in regions coding predomain, two affected the prodomain and nine others occurred in the mature domain. The novel lesions consisted in six missense mutations c.20T>C (p.L7P), c.494A>G (p.Q165R), c.580G>A (p.G194S), c.746T>C (p.I249T), c.749A>G (p.D250G), c.955G>T (p.G319C), two frameshifts c.60_61dupGA (p.I21RfsX29), c.282dupA (p.S95VfsX9) and a splicing mutation c.890G>A (r.785_890del). The six new missense mutations were examined by western blots of COS‐7 cells transfected with mutant CTSK genes. The L7P, occurring within the predicted hydrophobic domain of signal peptide, showed a significantly reduced expression level compared to the wild type control. These findings suggested that the mutation affected targeting and translocation of the nascent lysosomal protein across the endoplasmatic reticulum membrane. The novel amino acid changes were also modeled into the three‐dimensional structure that predicted incorrect protein folding for all of them. Molecular characterization of the patients is of particular value for genetic counseling of patients and their families as diagnosis of Pycnodysostosis based on enzyme assay is unpractical and thus not offered routinely. © 2007 Wiley‐Liss, Inc.     

Identification of seven novel SMPD1 mutations causing Niemann–Pick disease types A and B

Niemann–Pick disease (NPD) types A and B are autosomal, recessively inherited, lysosomal storage disorders caused by deficient activity of acid sphingomyelinase (E.C. 3.1.4.12) because of mutations in the sphingomyelin phosphodiesterase‐1 (SMPD1) gene. Here, we present the molecular analysis and clinical characteristics of 15 NPD type A and B patients. Sequencing the SMDP1 gene revealed eight previously described mutations and seven novel mutations including four missense [c.682T>C (p.Cys228Arg), c.1159T>C (p.Cys387Arg), c.1474G>A (p.Gly492Ser), and c.1795C>T (p.Leu599Phe)], one frameshift [c.169delG (p.Ala57Leufs*20)] and two splicing (c.316+1G>T and c.1341delG). The most frequent mutations were p.Arg610del (21%) and p.Gly247Ser (12%). Two patients homozygous for p.Arg610del and initially classified as phenotype B showed different clinical manifestations. Patients homozygous for p.Leu599Phe had phenotype B, and those homozygous for c.1341delG or c.316+1G>T presented phenotype A. The present results provide new insight into genotype/phenotype correlations in NPD and emphasize the difficulty of classifying patients into types A and B, supporting the idea of a continuum between these two classic phenotypes.     

Mutation spectra and founder effect of TMC1 in patients with non‐syndromic deafness in Xiamen area,China

To analyze the spectrum and founder effect of TMC1 mutations in patients with non‐syndromic deafness in the Xiamen area. Sporadic pedigrees were detected by targeted next‐generation sequencing, and 110 unrelated patients from Xiamen Special Education School were analyzed through Sanger sequencing for the TMC1 gene. In total, 53 SNPs were designed to analyze the haplotypes of the TMC1 c.2050G>C mutation. The probands of three families were found to be homozygous for TMC1 c.2050G>C, and their parents were all heterozygous for the TMC1 c.2050G>C mutation. In 110 unrelated patients from Xiamen Special Education School, four were found to carry compound heterozygotes of TMC1 c.2050G>C, which were compound heterozygotes of c.804G>A, c.1127T>C, c.1165C>T, and c.1396_1398delAAC, respectively. Three types of TMC1 polymorphisms (c.45C>T, c.1713C>T, c.2208+49C>T) and two heterozygotes of novel variants (c.1764‐4C>A, c.2073G>A[p.K691K]) were found in the remaining 100 patients. In total, four novel variants were detected in this study. These mutations and variants were not detected in 100 normal samples. The haplotypes of the probands of families with TMC1 c.2050G>C were identical. There were unique hotspots and spectra of TMC1 mutations in the Xiamen deaf population. Haplotype analysis is useful to understand the founder effect of the hot spot mutation.     

Founder Haplotype Analysis of Fanconi Anemia in the Korean Population Finds Common Ancestral Haplotypes for a FANCG Variant

A common ancestral haplotype is strongly suggested in the Korean and Japanese patients with Fanconi anemia (FA), because common mutations have been frequently found: c.2546delC and c.3720_3724delAAACA of FANCA; c.307+1G>C, c.1066C>T, and c.1589_1591delATA of FANCG. Our aim in this study was to investigate the origin of these common mutations of FANCA and FANCG. We genotyped 13 FA patients consisting of five FA‐A patients and eight FA‐G patients from the Korean FA population. Microsatellite markers used for haplotype analysis included four CA repeat markers which are closely linked with FANCA and eight CA repeat markers which are contiguous with FANCG. As a result, Korean FA‐A patients carrying c.2546delC or c.3720_3724delAAACA did not share the same haplotypes. However, three unique haplotypes carrying c.307+1G>C, c.1066C > T, or c.1589_1591delATA, that consisted of eight polymorphic loci covering a flanking region were strongly associated with Korean FA‐G, consistent with founder haplotypes reported previously in the Japanese FA‐G population. Our finding confirmed the common ancestral haplotypes on the origins of the East Asian FA‐G patients, which will improve our understanding of the molecular population genetics of FA‐G. To the best of our knowledge, this is the first report on the association between disease‐linked mutations and common ancestral haplotypes in the Korean FA population.     

Molecular genetic analyses of β-thalassemia in South India reveals rare mutations in the β-globin gene

-thalassemia is the most prevalent single-gene disorder. Since no viable forms of treatment are available, the best course is prevention through prenatal diagnosis. In the present study, the prevalence of -thalassemia was extensively investigated in the South Indian population, especially from the state of Andhra Pradesh. Screening for causal mutations was carried out on genomic DNA isolated from patient blood samples by using the routine reverse dot blot (RDB) and amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) techniques. DNA sequencing was performed wherever necessary. Among the nine mutations identified, four, including IVS-1-5 (G-C) (IVS1+5G>T), codon 41/42 (-TTCT) (c.124_127delTTCT), codon 15 (G-A) (c.47G>A), and HbS (sickle mutation) (c.20A>T) mutations, accounted for about 98% of the total positive cases. Two mutations viz. codon 8/9 (+G) (c.27_28insG) and HbE (codon 26 G-A) (c.79G>A) exhibited a very low frequency of occurrence, whereas the IVS-1-1 (G-T) (IVS1+1G>T) and the 619 bp deletion (c.366_494del) mutations were absent. We also identified certain rare mutations during the diagnostic evaluation. Gene sequencing confirmed the codon 30 (G-C) (c.92G>C) mutation and the rare codon 5 (-CT) (c.17_18delCT) and IVS-II-837 (T-G) (IVSII-14T>G) mutations. This is the first report of the IVS II 837 mutation in the Indian population. We also report a novel diagnostic application during RDB-based screening for the detection of the (c.92G>C) mutations. Such a comprehensive mutation screening is essential for prenatal diagnosis of -thalassemia and control of this highly prevalent monogenic disorder in the Indian population.     

Mutations of the EPHA2 receptor tyrosine kinase gene cause autosomal dominant congenital cataract

Congenital cataracts (CCs) are clinically and genetically heterogeneous. Mutations in the same gene may lead to CCs differing in inheritance, morphology and severity. Loci for autosomal dominant posterior polar CC and total CC have both been mapped to the chromosomal 1p36 region harboring the EPHA2 receptor tyrosine kinase gene. Here, we report mutations of EPHA2 in three CC families from different ancestral groups. In a Chinese family with posterior polar CC, we identified a missense mutation, c.2819C>T (p.T940I), replacing a critical amino acid that functions at the receptor oligomerization interface. In a British family with posterior polar CC and an Australian family with total CC, we found a frameshift mutation (c.2915_2916delTG) and a splicing mutation (c.2826‐9G>A), respectively. These two mutations are predicted to produce novel C‐terminal polypeptides with 39 identical amino acids. Yeast two‐hybrid analysis showed stronger interaction between the total CC‐associated mutant EPHA2 and low molecular weight protein‐tyrosine phosphatase, a negative regulator of EPHA2 signaling. Our results implicate the Eph‐ephrin signaling system in development of human cataract and provide a novel insight into the molecular mechanism underlying the pathogenesis of human CCs. © 2009 Wiley‐Liss, Inc.     

FANCA and FANCG are the major Fanconi anemia genes in the Korean population

Fanconi anemia (FA) is a rare disorder characterized by physical abnormalities, bone marrow failure (BMF), increased risk of malignancies, and cellular hypersensitivity to DNA cross‐linking agents. This study evaluated the genetic alterations in three major Fanconi genes (FANCA, FANCC, and FANCG) in 30 FA patients using multiplex ligation‐dependent probe amplification and direct sequencing. Thirteen BMF patients were genetically classified as FA‐A (n = 6, 46%) and FA‐G (n = 7, 54%). Four common founder mutations were identified and included two FANCA mutations (c.2546delC and c.3720_3724delAAACA) and two FANCG mutations (c.307+1G>C and c.1066C>T), which had previously been commonly observed in a Japanese FA population. We also detected four novel deleterious mutations: c.2778+1G>C and c.3627‐1G>A of FANCA, and c.1589_1591delATA and c.1761‐1G>A of FANCG. This study shows that mutations in FANCA and FANCG are common in Korean FA patients and the existence of four common founder mutations in an East Asian FA population. Mutation screening workflow that includes these common mutations may be useful in the creation of an international database, and to better understand the ethnic characteristics of FA.     

Novel WEE2 homozygous mutations c.1346C>T and c.949A>T identified in primary infertile women due to unexplained fertilization failure

The occurrence of unexplained fertilization failure can have profound psychological and financial consequences for couples struggling with infertility, and its pathogenesis remains unclear. Increasing evidence highlights genetic basis of unexplained fertilization failure occurrence. Here, we identified one novel homozygous nonsense mutation (c.949A>T), one novel homozygous missense mutation (c.1346C>T), and three reported homozygous mutations (c.585G>C, c.1006_1007insTA, c.1221G>A) in six unrelated probands, showing similar manifestations of unexplained fertilization failure. This finding expands the spectrum of WEE2 mutations, highlighting the critical role of WEE2 in fertilization process, and provides a basis for the prognostic value of testing for WEE2 mutations in primary infertile couples with unexplained fertilization failure.     

Glucose-6-phosphatase gene mutations in Taiwan Chinese patients with glycogen storage disease type Ia

Glycogen storage disease type Ia (GSD Ia) is caused by a deficiency of glucose-6-phosphatase (G6Pase) activity. Eighteen GSD Ia families were studied for G6Pase gene mutations. Thirty-two mutations were found in 36 GSD Ia chromosomes: 16 were 727 G→T (44.44%); 13 were R83H (327 G→T; 36.11%); 1 was 341delG; 1 was 933insAA; and 1 was 793 G→T. The 727 G→T and R83H mutations together accounted for 80.56% (29/36) of the GSD Ia chromosomes. These two mutations were easily examined by polymerase chain reaction-based methods, and the prenatal diagnosis of a non-affected fetus was successfully made. The 727 G→T mutation is the predominant mutation in Japanese GSD Ia patients, but is rarely seen in Western counties. The 727 G→T mutation is also the most prevalent mutation in Taiwan Chinese, although the incidence is not as high as in Japan. Received: January 4, 2000 / Accepted: February 28, 2000     

Prevalence of Mutations in Deafness‐Causing Genes in Cochlear Implanted Patients with Profound Nonsyndromic Sensorineural Hearing Loss in Shandong Province,China

The mutations of GJB2, SLC26A4, and mtDNA12SrRNA are the most common inherited causes of nonsyndromic sensorineural hearing loss (NSHL) in China, yet previous genetic screenings were mainly carried on patients with moderate‐to‐profound impairment. We aimed to detect the mutation frequencies in NSHL population within a more specified range of severity. Patients with profound NSHL who had undergone cochlear implantation in the Shandong Provincial Hospital (Shandong, China) were recruited. The majority (n = 472) were between 0.7 and 6 years old, and the remaining (n = 63) were between 6 and 70 years old. In total, 115 mutation alleles of the three genes were screened with SNP scan assay. Of the patients, 19.44% (104/535) were found to have GJB2 mutations, and the most common allele was c.235delC, followed by c.299_300delAT and c.109G>A. SLC26A4 mutations were detected in 13.46% patients (72/535), and the most common allele was c.919‐2A>G (IVS7‐2A>G), followed by c.1174A>T and c.2168A>G. Seven patients (1.31%) carried mutations in mtDNA12SrRNA, with the alleles of m.1555A>G and m.1494C>T. We found the allele frequency of c.109G>A (GJB2) was relatively lower in the profound NSHL population in comparison to the moderate‐to‐profound ones, and the c.1174A>T (SLC26A4) relatively higher. It suggests those mutations may be connected with the degree of deafness, which needs more observations and analyses to support.     

Pathogenic variants of DNAJC12 and evaluation of the encoded cochaperone as a genetic modifier of hyperphenylalaninemia

Biallelic variants of the gene DNAJC12, which encodes a cochaperone, were recently described in patients with hyperphenylalaninemia (HPA). This paper reports the retrospective genetic analysis of a cohort of unsolved cases of HPA. Biallelic variants of DNAJC12 were identified in 20 patients (generally neurologically asymptomatic) previously diagnosed with phenylalanine hydroxylase (PAH) deficiency (phenylketonuria [PKU]). Further, mutations of DNAJC12 were identified in four carriers of a pathogenic variant of PAH. The genetic spectrum of DNAJC12 in the present patients included four new variants, two intronic changes c.298‐2A>C and c.502+1G>C, presumably affecting the splicing process, and two exonic changes c.309G>T (p.Trp103Cys) and c.524G>A (p.Trp175Ter), classified as variants of unknown clinical significance (VUS). The variant p.Trp175Ter was detected in 83% of the mutant alleles, with 14 cases homozygous, and was present in 0.3% of a Spanish control population. Functional analysis indicated a significant reduction in PAH and its activity, reduced tyrosine hydroxylase stability, but no effect on tryptophan hydroxylase 2 stability, classifying the two VUS as pathogenic variants. Additionally, the effect of the overexpression of DNAJC12 on some destabilizing PAH mutations was examined and a mutation‐specific effect on stabilization was detected suggesting that the proteostasis network could be a genetic modifier of PAH deficiency and a potential target for developing mutation‐specific treatments for PKU.