Recurrent and founder mutations in the PMS2 gene

Germline mutations in PMS2 are associated with Lynch syndrome (LS), the most common known cause of hereditary colorectal cancer. Mutation detection in PMS2 has been difficult due to the presence of several pseudogenes, but a custom‐designed long‐range PCR strategy now allows adequate mutation detection. Many mutations are unique. However, some mutations are observed repeatedly across individuals not known to be related due to the mutation being either recurrent, arising multiple times de novo at hot spots for mutations, or of founder origin, having occurred once in an ancestor. Previously, we observed 36 distinct mutations in a sample of 61 independently ascertained Caucasian probands of mixed European background with PMS2 mutations. Eleven of these mutations were detected in more than one individual not known to be related and of these, six were detected more than twice. These six mutations accounted for 31 (51%) ostensibly unrelated probands. Here, we performed genotyping and haplotype analysis in four mutations observed in multiple probands and found two (c.137G>T and exon 10 deletion) to be founder mutations and one (c.903G>T) a probable founder. One (c.1A>G) could not be evaluated for founder mutation status. We discuss possible explanations for the frequent occurrence of founder mutations in PMS2.     

Identification of novel RMRP mutations and specific founder haplotypes in Japanese patients with cartilage-hair hypoplasia

Cartilage-hair hypoplasia (CHH), or metaphyseal dysplasia, McKusick type, is an autosomal recessive disease with diverse clinical manifestations. CHH is caused by mutations in RMRP (ribonuclease mitochondrial RNA processing), the gene encoding the RNA component of the ribonucleoprotein complex RNase MRP. A common founder mutation, 70A>G has been reported in the Finnish and Amish populations. We screened 11 Japanese patients with CHH for RMRP mutations and identified mutations in five probands, including three novel mutations (16-bp dup at +1, 168G>A, and 217C>T). All patients were compound heterozygotes for an insertion or duplication in the promoter or 5′-transcribed regions and a point mutation in the transcribed region. Two recurrent mutations were unique to the Japanese population: a 17-bp duplication at +3 and 218A>G. Haplotype analysis revealed that the two mutations common in Japanese individuals were contained within distinct haplotypes. Through this analysis, we have identified a unique mutation spectrum and founder mutations in the Japanese population.Yuichiro Hirose and Eiji Nakashima contributed equally to this work.     

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.     

Pathogenic Mitochondrial tRNA Point Mutations: Nine Novel Mutations Affirm Their Importance as a Cause of Mitochondrial Disease

Mutations in the mitochondrial genome, and in particular the mt‐tRNAs, are an important cause of human disease. Accurate classification of the pathogenicity of novel variants is vital to allow accurate genetic counseling for patients and their families. The use of weighted criteria based on functional studies—outlined in a validated pathogenicity scoring system—is therefore invaluable in determining whether novel or rare mt‐tRNA variants are pathogenic. Here, we describe the identification of nine novel mt‐tRNA variants in nine families, in which the probands presented with a diverse range of clinical phenotypes including mitochondrial encephalomyopathy, lactic acidosis, and stroke‐like episodes, isolated progressive external ophthalmoplegia, epilepsy, deafness and diabetes. Each of the variants identified (m.4289T>C, MT‐TI; m.5541C>T, MT‐TW; m.5690A>G, MT‐TN; m.7451A>T, MT‐TS1; m.7554G>A, MT‐TD; m.8304G>A, MT‐TK; m.12206C>T, MT‐TH; m.12317T>C, MT‐TL2; m.16023G>A, MT‐TP) was present in a different tRNA, with evidence in support of pathogenicity, and where possible, details of mutation transmission documented. Through the application of the pathogenicity scoring system, we have classified six of these variants as “definitely pathogenic” mutations (m.5541C>T, m.5690A>G, m.7451A>T, m.12206C>T, m.12317T>C, and m.16023G>A), whereas the remaining three currently lack sufficient evidence and are therefore classed as ‘possibly pathogenic’ (m.4289T>C, m.7554G>A, and m.8304G>A).     

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.     

Sequestosome-1 (SQSTM1) sequence variants in ALS cases in the UK: prevalence and coexistence of SQSTM1 mutations in ALS kindred with PDB

Mutations in the SQSTM1 gene have been reported to be associated with amyotrophic lateral sclerosis (ALS). We sought to determine the frequency of these mutations in a UK familial ALS (FALS) cohort. Sequences of all eight exons of the SQSTM1 gene were analysed in index cases from 61 different FALS kindred lacking known FALS mutations. Six exonic variants c.463G>A, p.(Glu155Lys), c.822G>C, p.(Glu274Asp), c.888G>T, p.(=), c.954C>T, p.(=), c.1038G>A, p.(=) and c.1175C>T, p.(Pro392Leu) were identified in five FALS index cases, three of which were non-synonymous and three were synonymous. One index case harboured three variants (c.822G>C, c.888G>T and c.954C>T), and a second index case harboured two variants (c.822G>C and c.954C>T). Only the p.(Pro392Leu) and p.(Glu155Lys) mutations were predicted to be pathogenic. In one p.(Pro392Leu) kindred, the carrier developed both ALS and Paget''s disease of bone (PDB), and, in the p.(Glu155Lys) kindred, the father of the proband developed PDB. All p.(Pro392Leu) carriers were heterozygous for a previously reported founder haplotype for PDB, where this mutation has an established causal effect. The frequency of the p.(Pro392Leu) mutation in this UK FALS cohort was 2.3% and 0.97% overall including three previously screened FALS cohorts. Our results confirm the presence of the p.(Pro392Leu) SQSTM1 mutation in FALS. This mutation is the most common SQSTM1 mutation found in ALS to date, and a likely pathogenicity is supported by having an established causal role in PDB. The occurrence of the same mutation in ALS and PDB is indicative of a common pathogenic pathway that converges on protein homeostasis.     

Genetic and bioinformatics analysis of four novel GCK missense variants detected in Caucasian families with GCK‐MODY phenotype

Heterozygous loss‐of‐function mutations in the glucokinase (GCK) gene cause maturity‐onset diabetes of the young (MODY) subtype GCK (GCK‐MODY/MODY2). GCK sequencing revealed 16 distinct mutations (13 missense, 1 nonsense, 1 splice site, and 1 frameshift‐deletion) co‐segregating with hyperglycaemia in 23 GCK‐MODY families. Four missense substitutions (c.718A>G/p.Asn240Asp, c.757G>T/p.Val253Phe, c.872A>C/p.Lys291Thr, and c.1151C>T/p.Ala384Val) were novel and a founder effect for the nonsense mutation (c.76C>T/p.Gln26*) was supposed. We tested whether an accurate bioinformatics approach could strengthen family‐genetic evidence for missense variant pathogenicity in routine diagnostics, where wet‐lab functional assays are generally unviable. In silico analyses of the novel missense variants, including orthologous sequence conservation, amino acid substitution (AAS)‐pathogenicity predictors, structural modeling and splicing predictors, suggested that the AASs and/or the underlying nucleotide changes are likely to be pathogenic. This study shows how a careful bioinformatics analysis could provide effective suggestions to help molecular‐genetic diagnosis in absence of wet‐lab validations.     

Clinical and molecular analysis in Papillon–Lefèvre syndrome

Papillon–Lefèvre syndrome (PLS; MIM#245000) is a rare recessive autosomal disorder characterized by palmar and plantar hyperkeratosis, and aggressively progressing periodontitis leading to premature loss of deciduous and permanent teeth. PLS is caused by loss‐of‐function mutations in the CTSC gene, which encodes cathepsin C. PLS clinical expressivity is highly variable and no consistent genotype–phenotype correlation has been demonstrated yet. Here we report the clinical and genetic features of five PLS patients presenting a severe periodontal breakdown in primary and permanent dentition, hyperkeratosis over palms and soles, and recurrent sinusitis and/or tonsillitis. Mutation analysis revealed two novel homozygous recessive mutations (c.947T>C and c.1010G>C) and one previous described homozygous recessive mutation (c.901G>A), with parents carrying them in heterozygous, in three families (four patients). The fourth family presented with the CTSC c.628C>T mutation in heterozygous, which was inherited maternally. Patient carrying the CTSC c.628C>T mutation featured classical PLS phenotype, but no PLS clinical characteristics were found in his carrier mother. All mutations were found to affect directly (c.901G>A, c.947T>C, and c.1010G>C) or indirectly (c.628C>T, which induces a premature termination) the heavy chain of the cathepsin C, the region responsible for activation of the lysosomal protease. Together, these findings indicate that both homozygous and heterozygous mutations in the cathepsin C heavy chain domain may lead to classical PLS phenotype, suggesting roles for epistasis or gene–environment interactions on determination of PLS phenotypes.     

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.     

Identification of TMC1 as a relatively common cause for nonsyndromic hearing loss in the Saudi population

Hearing loss (HL) is the most common sensory disorder worldwide and genetic factors contribute to approximately half of congenital HL cases. HL is subject to extensive genetic heterogeneity, rendering molecular diagnosis difficult. Mutations of the transmembrane channel‐like 1 (TMC1) gene cause hearing defects in humans and mice. The precise function of TMC1 protein in the inner ear is unknown, although it is predicted to be involved in functional maturation of cochlear hair cells. TMC1 mutations result in autosomal recessive (DFNB7/11) and sometimes dominant (DFNA36) nonsyndromic HL. Mutations in TMC1 are responsible for a significant portion of HL, particularly in consanguineous populations. To evaluate the importance of TMC1 mutations in the Saudi population, we used a combination of autozygome‐guided candidate gene mutation analysis and targeted next generation sequencing in 366 families with HL previously shown to lack mutations in GJB2. We identified 12 families that carried five causative TMC1 mutations; including three novel (c.362+3A > G; c.758C > T [p.Ser253Phe]; c.1396_1398delACC [p.Asn466del]) and two reported mutations (c.100C > T [p.Arg34Ter]; c.1714G > A [p.Asp572Asn]). Each of the identified recessive mutation was classified as severe, by both age of onset and severity of HL. Similarly, consistent with the previously reported dominant variant p.Asp572Asn, the HL phenotype was progressive. Eight families in our cohort were found to share the pathogenic p.Arg34Ter mutation and linkage disequilibrium was observed between p.Arg34Ter and SNPs investigated. Our results indicate that TMC1 mutations account for about 3.3% (12/366) of Saudi HL cases and that the recurrent TMC1 mutation p.Arg34Ter is likely to be a founder mutation.     

Hereditary angioedema in a Jordanian family with a novel missense mutation in the C1-inhibitor N-terminal domain

Hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) is an autosomal dominant disease caused by mutations in the SERPING1 gene. A Jordanian family, including 14 individuals with C1-INH-HAE clinical symptoms, was studied. In the propositus and his parents, SERPING1 had four mutations leading to amino acid substitutions. Two are known polymorphic variants (c.167T>C; p.Val34Ala and c.1438G>A; p.Val458Met), the others are newly described. One (c.203C>T; p.Thr46Ile) is located in the N-terminal domain of the C1-inhibitor protein and segregates with angioedema symptoms in the family. The other (c.800C>T; p.Ala245Val) belongs to the serpin domain, and derives from the unaffected father. DNA from additional 24 family members were screened for c.203C>T mutation in the target gene. All individuals heterozygous for the c.203C>T mutation had antigenic and functional plasma levels of C1-inhibitor below 50% of normal, confirming the diagnosis of type I C1-INH-HAE. Angioedema symptoms were present in 14 of 16 subjects carrier for the c.203T allele. Among these subjects, those carrying the c.800T variation had more severe and frequent symptoms than subjects without this mutation. This family-based study provides the first evidence that multiple amino acid substitutions in SERPING1 could influence C1-INH-HAE phenotype.     

De novo mutations in FLNC leading to early‐onset restrictive cardiomyopathy and congenital myopathy

Mutations in FLNC for a long time are known in connection to neuromuscular disorders and only recently were described in association with various cardiomyopathies. Here, we report a new clinical phenotype of filaminopathy in four unrelated patients with early‐onset restrictive cardiomyopathy (RCM) in combination with congenital myopathy due to FLNC mutations (NM_001458.4:c.3557C>T, p.A1186V, rs1114167361 in three probands and c.[3547G>C; 3548C>T], p.A1183L, rs1131692185 in one proband). In all cases, concurrent myopathy was confirmed by neurological examination, electromyography, and morphological studies. Three of the patients also presented with arthrogryposis. The pathogenicity of the described missense variants was verified by cellular and morphological studies and by in vivo modeling in zebrafish. Combination of in silico and experimental approaches revealed that FLNC missense variants localized in Ig‐loop segments often lead to development of RCM. The described FLNC mutations associated with early‐onset RCMP extend cardiac spectrum of filaminopathies and facilitate the differential diagnosis of restrictive cardiac phenotype associated with neuromuscular involvement in children.     

Allele-specific PCR and Next-generation sequencing based genetic screening for Congenital Adrenal Hyperplasia in India

Genetic screening of Congenital Adrenal Hyperplasia (CAH) is known to be challenging due to the complexities in CYP21A2 genotyping and has not been the first-tier diagnostic tool in routine clinical practice. Also, with the advent of massive parallel sequencing technology, there is a need for investigating its utility in screening extended panel of genes implicated in CAH. In this study, we have established and utilized an Allele-Specific Polymerase Chain Reaction (ASPCR) based approach for screening eight common mutations in CYP21A2 gene followed by targeted Next Generation Sequencing (NGS) of CYP21A2, CYP11B1, CYP17A1, POR, and CYP19A1 genes in 72 clinically diagnosed CAH subjects from India. Through these investigations, 88.7% of the subjects with 21 hydroxylase deficiency were positive for eight CYP21A2 mutations with ASPCR. The targeted NGS assay was sensitive to pick up all the mutations identified by ASPCR. Utilizing NGS in subjects negative for ASPCR, five study subjects were homozygous positive for other CYP21A2 variants: one with a novel c.1274G>T, three with c.1451G>C and one with c.143A>G variant. One subject was compound heterozygous for c.955C>T and c.1042G>A variants identified using ASPCR and NGS. One subject suspected for a Simple Virilizing (SV) 21 hydroxylase deficiency was positive for a CYP19A1:c.1142A>T variant. CYP11B1 variants (c.1201-1G>A, c.1200+1del, c.412C>T, c.1024C>T, c.1012dup, c.623G>A) were identified in all six subjects suspected for 11 beta-hydroxylase deficiency. The overall mutation positivity was 97.2%. Our results suggest that ASPCR followed by targeted NGS is a cost-effective and comprehensive strategy for screening common CYP21A2 mutations and the CAH panel of genes in a clinical setting.     

Novel SLC5A2 Variants Contribute to Renal Glucosuria in Chinese Families: Abnormal Expression and Dysfunction of Variant SLC5A2

Familial renal glucosuria (FRG) is characterized by persistent glucosuria despite normal serum glucose and the absence of overt tubular dysfunction. Variants in solute carrier family 5 (sodium–glucose cotransporter), member 2 (SLC5A2) have been reported in FRG patients. However, the functional and expression‐related consequences of such variants have been scarcely investigated. In the current study, we studied five FRG families and identified six missense mutations, including four novel variants (c.1051T>C/.(C351R), c.1400T>C/p.(V467A), c.1420G>C/p.(A474P), c.1691G>A/p.(R564Q); RNA not analyzed) and two variants that had been previously reported (c.294C>A/p.(F98L), c.736C>T/p.(P246S); RNA not analyzed). The probands were either heterozygous or compound heterozygous for SLC5A2 variants and had glucosuria of 5.9%–19.6 g/day. Human 293 cells were transfected with plasmid constructs to study the expression and function of SLC5A2 variants in vitro. Western blotting revealed that the expression levels of SLC5A2–351R‐GFP, SLC5A2–467A‐GFP, SLC5A2–474P‐GFP, and SLC5A2–564Q‐GFP were significantly decreased compared with wild‐type SLC5A2‐GFP (37%–55%). Confocal microscopy revealed that three variants (c.1400T>C, c.1420G>C, c.1691G>A) resulted in a loss of the punctate membrane pattern typical of wild‐type SLC5A2. All variants had a significantly lower transport capacity in than the wild‐type control. The current study provides a starting point to further investigate the molecular mechanism of SLC5A2 in FRG families and provides functional clues for antidiabetes drugs.     

The nonsense mutation MSH2 c.2152C>T shows a founder effect in Portuguese Lynch syndrome families

The mutational spectrum of the MMR genes is highly heterogeneous, but specific mutations are observed at high frequencies in well‐defined populations or ethnic groups, due to founder effects. The MSH2 mutation c.2152C>T, p.(Gln718*), has occasionally been described in Lynch families worldwide, including in Portuguese Lynch syndrome families. During genetic testing for Lynch syndrome at the Portuguese Oncology Institutes of Porto and Lisbon, this mutation was identified in 28 seemingly unrelated families. In order to evaluate if this alteration is a founder mutation, haplotype analysis using microsatellite and SNP markers flanking the MSH2 gene was performed in the 28 probands and 87 family members. Additionally, the geographic origin of these families was evaluated and the age of the mutation estimated. Twelve different haplotypes were phased for 13 out of the 28 families and shared a conserved region of ～3.6 Mb. Based on the mutation and recombination events observed in the microsatellite haplotypes and assuming a generation time of 25 years, the age estimate for the MSH2 mutation was 273 ± 64 years. The geographic origins of these families were mostly from the Northern region of Portugal. Concluding, these results suggest that the MSH2 c.2152C>T alteration is a founder mutation in Portugal with a relatively recent origin. Furthermore, its high proportion indicates that screening for this mutation as a first step, together with the previously reported Portuguese founder mutations, may be cost‐effective in genetic testing of Lynch syndrome suspects of Portuguese ancestry.     

The homozygous R504C mutation in MTO1 gene is responsible for ONCE syndrome

We report clinical and biochemical finding from three unrelated patients presenting ONCE (Optic Neuropathy, Cardiomyopathy and Encephalopathy with lactic acidosis and combined oxidative phosphorylation deficiency) syndrome. Whole‐exome sequencing (WES) of the three patients and the healthy sister of one of them was used to identify the carry gene. Clinical and biochemical findings were used to filter variants, and molecular, in silico and genetic studies were performed to characterize the candidate variants. Mitochondrial DNA (mtDNA) defects involving mutations, deletions or depletion were discarded, whereas WES uncovered a double homozygous mutation in the MTO1 gene (NM_001123226:c.1510C>T, p.R504C, and c.1669G>A, p.V557M) in two of the patients and the homozygous mutation p.R504C in the other. Therefore, our data confirm p.R504C as pathogenic mutation responsible of ONCE syndrome, and p.V557M as a rare polymorphic variant.     

Comprehensive functional assessment of MLH1 variants of unknown significance

Lynch syndrome is associated with germline mutations in DNA mismatch repair (MMR) genes. Up to 30% of DNA changes found are variants of unknown significance (VUS). Our aim was to assess the pathogenicity of eight MLH1 VUS identified in patients suspected of Lynch syndrome. All of them are novel or not previously characterized. For their classification, we followed a strategy that integrates family history, tumor pathology, and control frequency data with a variety of in silico and in vitro analyses at RNA and protein level, such as MMR assay, MLH1 and PMS2 expression, and subcellular localization. Five MLH1 VUS were classified as pathogenic: c.[248G>T(;)306G>C], c.[780C>G;788A>C], and c.791‐7T>A affected mRNA processing, whereas c.218T>C (p.L73P) and c.244G>A (p.T82A) impaired MMR activity. Two other VUS were considered likely neutral: the silent c.702G>A variant did not affect mRNA processing or stability, and c.974G>A (p.R325Q) did not influence MMR function. In contrast, variant c.25C>T (p.R9W) could not be classified, as it associated with intermediate levels of MMR activity. Comprehensive functional assessment of MLH1 variants was useful in their classification and became relevant in the diagnosis and genetic counseling of carrier families. Hum Mutat 33:1576–1588, 2012. © 2012 Wiley Periodicals, Inc.     

New EPCAM founder deletion in Polish population

It is well known that founder mutations associated with cancer risk have useful implications for molecular diagnostics. We report the presence of a founder mutation in EPCAM involved in the etiology of Lynch syndrome (LS). The mutation extends nearly 8.7 kb (c.858 + 2478_*4507del) and is shared by 8 Polish families. Family members suffered almost exclusively from colorectal cancer; however, pancreatic and gastric cancers were also apparent. Next to mutations c. 2041G>A in MLH1 gene and c.942+3A>T in MSH2, the deletion mutation encompassing EPCAM is one of the most common causative changes responsible for LS in Poland.