Classical and Atypical Fibrodysplasia Ossificans Progressiva in India

Fibrodysplasia Ossificans Progressiva (FOP) is a rare debilitating disorder characterized by congenital deformity of the great toes from infancy and postnatal heterotopic ossification. Activating mutations in the activin A receptor type 1 (ACVR1) gene are responsible for the disease. The most common allelic variant leading to FOP is c.617 G>A; p.R206H, however, other alleles have been reported with atypical phenotypes. We report 14 cases presenting to a referral institution in South India over a 3‐year period. The patients were clinically diagnosed based on foot abnormality or abnormal ectopic ossification and were screened for ACVR1. The genetic analysis of ACVR1 identified the recurrent allelic variant in 12 of 14 patients. One of the remaining patients had a previously reported allele c.1067G>A; p.G356D in the 9th exon and the second allele c.983G>A; p.G328E in the 8th exon of ACVR1. The most common recurrent allele c.617 G>A; p.R206H is also the most common in Indian patients with FOP.     

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.     

Carrier screening of RTEL1 mutations in the Ashkenazi Jewish population

Hoyeraal–Hreidarsson syndrome (HH) is a clinically severe variant of dyskeratosis congenita (DC), characterized by cerebellar hypoplasia, microcephaly, intrauterine growth retardation, and severe immunodeficiency in addition to features of DC. Germline mutations in the RTEL1 gene have recently been identified as causative of HH. In this study, the carrier frequency for five RTEL1 mutations that occurred in individuals of Ashkenazi Jewish descent was investigated in order to advise on including them in existing clinical mutation panels for this population. Our screening showed that the carrier frequency for c.3791G>A (p.R1264H) was higher than expected, 1% in the Ashkenazi Orthodox and 0.45% in the general Ashkenazi Jewish population. Haplotype analyses suggested the presence of a common founder. We recommend that the c.3791G>A RTEL1 mutation be considered for inclusion in carrier screening panels in the Ashkenazi population.     

Split hand/foot malformation associated with 20p12.1 deletion: A case report

Split hand/foot malformation (SHFM) or ectrodactyly is a rare congenital disorder affecting limb development characterized by clinical and genetic heterogeneity. SHFM is usually inherited as an autosomal dominant trait with incomplete penetrance. Isolated and syndromic forms are described. The extent of associated malformations is highly variable and multiple syndromes with clinical and genetic overlap have been described. We report here a 28 year-old man presenting with SHFM, sparse hair and widespread freckles. Array-CGH identified a 450 kb de novo 20p12.1 microdeletion encompassing three exons (exon 6 to 8) of MACROD2. Although MACROD2 mutations have not been associated with limb malformation until now, it is located next to KIF16B, which is involved in fibroblast growth factor receptor (FGFR) signaling. Additionally, the deletion encompassed a histone modification H3K27ac mark, known as a provider of quantitative readout of promoter and enhancer activity during human limb development. Altogether, these findings suggest that the 20p12.1 CNV is causative of SHFM in the present case through disturbance of regulatory elements functioning.     

Hermansky–Pudlak syndrome type 2: Aberrant pre‐mRNA splicing and mislocalization of granule proteins in neutrophils

Hermansky–Pudlak syndrome type 2 (HPS2) is a syndrome caused by mutations in the beta‐3A subunit of the adaptor protein (AP)‐3 complex (AP3B1 gene). We describe five unreported cases with four novel mutations, one of which caused aberrant pre‐mRNA splicing. A point mutation c.2702C>G in exon 23 of the AP3B1 gene caused deletion of 112 bp in the mRNA in two siblings. This mutation activates a cryptic donor splice site that overrules the wild‐type donor splice site of this exon. Three other novel mutations in AP3B1 were identified, that is, a nonsense mutation c.716G>A (p.Trp239Ter), a 1‐bp and a 4‐bp deletion c.177delA and c.1839_1842delTAGA, respectively, both causing frameshift and premature termination of translation. Mass spectrometry in four of these HPS2 patients demonstrated the (near) absence of all AP‐3 complex subunits. Immunoelectron microscopy on the neutrophils of two of these patients showed abnormal granule formation. We found clear mislocalization of myeloperoxidase in the neutrophils even though the content of this protein but not the activity seemed to be present at normal levels. In sum, HPS2 is the result of the absence of the entire AP‐3 complex, which results in severe neutropenia with a defect in granule formation as the major hematological finding.     

Novel LDLR Variants in Patients with Familial Hypercholesterolemia: In Silico Analysis as a Tool to Predict Pathogenic Variants in Children and Their Families

Familial hypercholesterolemia (FH) is an autosomal dominant disease with a frequency of 1:500 in its heterozygous form. To date, mutations in the low‐density lipoprotein receptor gene (LDLR) are the only identified causes of FH in the Greek population, causing high levels of low‐density lipoprotein (LDL) and total cholesterol and premature atherosclerosis. The Greek FH population is genetically homogeneous, but most previous studies screened for the most common mutations only. The study aimed to characterize and assess novel LDLR variants. LDLR was examined by whole‐gene DNA sequencing in 561 FH patients from 262 families of Greek origin. Novel LDLR variants were analyzed in silico using various software predicting pathogenicity and changes in protein stability. Twelve novel LDLR variants were identified, six of which are putative disease‐causing variants: c.977C>G in exon 7, c.1124A>C in exon 8, c.1381G>T in exon 10, c.628_643dup{636del}, c.661–673dup in exon 4, and 13 c.1987+1_+33del in intron 13. All six putative variants were confirmed in the hypercholesterolemic members of the family. The results show that in silico analysis is a valuable tool to predict potential pathogenicity of novel variants, especially for populations that have not been extensively studied. The identification of novel pathogenic variants will facilitate the molecular diagnosis of FH from early childhood.     

Ten novel HMGCL mutations in 24 patients of different origin with 3‐hydroxy‐3‐methyl‐glutaric aciduria

3‐Hydroxy‐3‐methylglutaric aciduria is a rare autosomal recessive genetic disorder that affects ketogenesis and L‐leucine catabolism. The clinical acute symptoms include vomiting, convulsions, metabolic acidosis, hypoketotic hypoglycaemia and lethargy. To date, 33 mutations in 100 patients have been reported in the HMGCL gene. In this study 10 new mutations in 24 patients are described. They include: 5 missense mutations: c.109G>A, c.425C>T, c.521G>A, c.575T>C and c.598A>T, 2 nonsense mutations: c.242G>A and c.559G>T, one small deletion: c.853delC, and 2 mutations in intron regions: c.497+4A>G and c.750+1G>A. Two prevalent mutations were detected, 109G>T (E37X) in 38% of disease alleles analyzed and c.504_505delCT in 10% of them. Although patients are mainly of European origin (71%) and mostly Spanish (54%), the group is ethnically diverse and includes, for the first time, patients from Pakistan, Palestine and Ecuador. We also present a simple, efficient method to express the enzyme and we analyze the possible functional effects of missense mutations. The finding that all identified missense mutations cause a >95% decrease in the enzyme activity, indicates that the disease appears only in very severe genotypes.” © 2009 Wiley‐Liss, Inc.     

Ex vivo splicing assays of mutations at noncanonical positions of splice sites in USHER genes

Molecular diagnosis in Usher syndrome type 1 and 2 patients led to the identification of 21 sequence variations located in noncanonical positions of splice sites in MYO7A, CDH23, USH1C, and USH2A genes. To establish experimentally the splicing pattern of these substitutions, whose impact on splicing is not always predictable by available softwares, ex vivo splicing assays were performed. The branch‐point mapping strategy was also used to investigate further a putative branch‐point mutation in USH2A intron 43. Aberrant splicing was demonstrated for 16 of the 21 (76.2%) tested sequence variations. The mutations resulted more frequently in activation of a nearby cryptic splice site or use of a de novo splice site than exon skipping (37.5%). This study allowed the reclassification as splicing mutations of one silent (c.7872G>A (p.Glu2624Glu) in CDH23) and four missense mutations (c.2993G>A (p.Arg998Lys) in USH2A, c.592G>A (p.Ala198Thr), c.3503G>C [p.Arg1168Pro], c.5944G>A (p.Gly1982Arg) in MYO7A), whereas it provided clues about a role in structure/function in four other cases: c.802G>A (p.Gly268Arg), c.653T>A (p.Val218Glu) (USH2A), and c.397C>T (p.His133Tyr), c.3502C>T (p.Arg1168Trp) (MYO7A). Our data provide insights into the contribution of splicing mutations in Usher genes and illustrate the need to define accurately their splicing outcome for diagnostic purposes. Hum Mutat 31:1–9, 2010. © 2010 Wiley‐Liss, Inc.     

Missense mutations in the AFG3L2 proteolytic domain account for ∼1.5% of European autosomal dominant cerebellar ataxias

Spinocerebellar ataxia type 28 is an autosomal dominant form of cerebellar ataxia (ADCA) caused by mutations in AFG3L2, a gene that encodes a subunit of the mitochondrial m‐AAA protease. We screened 366 primarily Caucasian ADCA families, negative for the most common triplet expansions, for point mutations in AFG3L2 using DHPLC. Whole‐gene deletions were excluded in 300 of the patients, and duplications were excluded in 129 patients. We found six missense mutations in nine unrelated index cases (9/366, 2.6%): c.1961C>T (p.Thr654Ile) in exon 15, c.1996A>G (p.Met666Val), c.1997T>G (p.Met666Arg), c.1997T>C (p.Met666Thr), c.2011G>A (p.Gly671Arg), and c.2012G>A (p.Gly671Glu) in exon 16. All mutated amino acids were located in the C‐terminal proteolytic domain. In available cases, we demonstrated the mutations segregated with the disease. Mutated amino acids are highly conserved, and bioinformatic analysis indicates the substitutions are likely deleterious. This investigation demonstrates that SCA28 accounts for ～3% of ADCA Caucasian cases negative for triplet expansions and, in extenso, to ～1.5% of all ADCA. We further confirm both the involvement of AFG3L2 gene in SCA28 and the presence of a mutational hotspot in exons 15–16. Screening for SCA28, is warranted in patients who test negative for more common SCAs and present with a slowly progressive cerebellar ataxia accompanied by oculomotor signs. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

Gene Conversion Between Cationic Trypsinogen (PRSS1) and the Pseudogene Trypsinogen 6 (PRSS3P2) in Patients with Chronic Pancreatitis

Mutations of the human cationic trypsinogen gene (PRSS1) are frequently found in association with hereditary pancreatitis. The most frequent variants p.N29I and p.R122H are recognized as disease‐causing mutations. Three pseudogene paralogs in the human trypsinogen family, including trypsinogen 6 (PRSS3P2), carry sequence variations in exon 3 that mimic the p.R122H mutation. In routine genetic testing of patients with chronic pancreatitis, we identified in two unrelated individuals similar gene conversion events of 24–71 nucleotides length between exon 3 of the PRSS1 (acceptor) and PRSS3P2 (donor) genes. The converted allele resulted in three nonsynonymous alterations c.343T>A (p.S115T), c.347G>C (p.R116P), and c.365_366delinsAT (p.R122H). Functional analysis of the conversion triple mutant revealed markedly increased autoactivation resulting in high and sustained trypsin activity in the presence of chymotrypsin C. This activation phenotype was identical to that of the p.R122H mutant. In addition, cellular secretion of the triple mutant from transfected HEK 293T cells was increased about twofold and this effect was attributable to mutation p.R116P. Our observations confirm and extend the notion that recombination events between members of the trypsinogen family can generate high‐risk PRSS1 alleles. The pathogenic phenotype of the novel conversion is explained by a unique combination of increased trypsinogen activation and secretion.     

Ethnic differences in GRHPR mutations in patients with primary hyperoxaluria type 2

The objective of this study was to investigate ethnic differences in the glyoxylate reductase/hydroxypyruvate reductase (GRHPR) gene in patients with primary hyperoxaluria type 2 (PH2). GRHPR was genotyped in Japanese patients with PH2 and all GRHPR mutations described to date were reviewed in terms of geographic and ethnic association. We identified a novel mutation, a two‐nucleotide deletion (c.248_249delTG) in exon 3 creating a premature ‘stop’ at codon 91. Also, we found that the c.864_865delTG mutation was associated with the rs35891798 single‐nucleotide polymorphism. The allelic frequencies of the c.103delG, c.494G>A, c.403_404+2 delAAGT, and c.864_865delTG mutations in PH2 patients were 37.8%, 15.6%, 10.0%, and 10.0%, respectively. All patients with the c.103delG mutation were Caucasian. Patients with the c.494G>A mutation and 78% (7/9) of those with the c.403_404+2 delAAGT mutation were from the Indian subcontinent, whereas those with the c.864_865delTG mutation were Chinese or Japanese. Molecular analysis of GRHPR of four Japanese PH2 patients identified a novel mutation (c.248_249delTG in exon 3). Caucasians with PH2 should be screened for the c.103delG mutation; patients from the Indian subcontinent for c.494G>A; and patients of East Asian origin (particularly) for c.864_865delTG. The prevalence of the latter mutation in PH2 patients from East Asia was 75.0%.     

Novel synonymous and missense variants in FGFR1 causing Hartsfield syndrome

Hartsfield syndrome is a rare clinical entity characterized by holoprosencephaly and ectrodactyly with the variable feature of cleft lip/palate. In addition to these symptoms patients with Hartsfield syndrome can show developmental delay of variable severity, isolated hypogonadotropic hypogonadism, central diabetes insipidus, vertebral anomalies, eye anomalies, and cardiac malformations. Pathogenic variants in FGFR1 have been described to cause phenotypically different FGFR1‐related disorders such as Hartsfield syndrome, hypogonadotropic hypogonadism with or without anosmia, Jackson–Weiss syndrome, osteoglophonic dysplasia, Pfeiffer syndrome, and trigonocephaly Type 1. Here, we report three patients with Hartsfield syndrome from two unrelated families. Exome sequencing revealed two siblings harboring a novel de novo heterozygous synonymous variant c.1029G>A, p.Ala343Ala causing a cryptic splice donor site in exon 8 of FGFR1 likely due to gonadal mosaicism in one parent. The third case was a sporadic patient with a novel de novo heterozygous missense variant c.1868A>G, p.(Asp623Gly).     

Novel mutations in the HPS1 gene among Puerto Rican patients

Carmona‐Rivera C, Hess RA, O’Brien K, Golas G, Tsilou E, White JG, Gahl WA, Huizing M. Novel mutations in the HPS1 gene among Puerto Rican patients. Hermansky‐Pudlak syndrome (HPS) is a disorder of oculocutaneous albinism (OCA) and platelet storage pool deficiency. Eight different disease‐causing genes have been identified, whose gene products are thought to be involved in the biogenesis of lysosome‐related organelles. HPS type 1 (HPS‐1) is the most common HPS subtype in Puerto Rico, with a frequency of 1:1800 in the northwest of the island due to a founder mutation, i.e. a 16‐bp duplication in exon 15 of the HPS1 gene (c.1472_1487dup16; p.H497QfsX90). We identified three Puerto Rican HPS‐1 patients who carried compound heterozygous HPS1 mutations. One patient was heterozygous for c.937G>A, causing a missense mutation (p.G313S) at the 3′ splice junction of exon 10. This mutation resulted in activation of a cryptic intronic splice site causing an aberrantly spliced HPS1 mRNA that included 144‐bp of intronic sequence, producing 11 novel amino acids followed by a stop codon. The other two patients were heterozygous for the previously reported c.972delC in HPS1, resulting in a frameshift and a premature stop codon (p.M325WfsX6). These findings indicate that, among Puerto Ricans, other HPS1 mutations apart from the 16‐bp duplication should be considered in the analysis of this population.     

Severe and mild phenotypes in Pfeiffer syndrome with splice acceptor mutations in exon IIIc of FGFR2

Pfeiffer syndrome is clinically and genetically heterogeneous. Three clinical subtypes have been delineated based on the severity of acrocephalysyndactyly and associated manifestations. Severe cases are usually sporadic and caused by a number of different mutations in exons IIIa and IIIc of the fibroblast growth factor receptor 2 (FGFR2) gene. Mild cases are either sporadic or familial and are caused by mutations in FGFR2 or FGFR1, respectively. We report on two individuals with different novel de novo mutations in FGFR2. The first is a 17‐year‐old male who has a severe phenotype, within the spectrum of subtype 1 including severe ocular proptosis, elbow ankylosis, visceral anomalies, and normal intelligence. This patient was found to have a novel complex mutation at the 3′ acceptor site of exon IIIc of FGFR2, denoted as C952‐3 del10insACC. The other patient, a 2‐year‐old female, has a mild phenotype, typical of the classic subtype 1 including brachycephaly with coronal synostosis and hypertelorism. She was also found to have a mutation at the 3′ acceptor site (the same splice site) of exon IIIc of FGFR2, a point mutation designated as 952‐1G→A. Speculation on the molecular mechanisms that cause severe and mild phenotypes is presented in relation to these two cases. © 2001 Wiley‐Liss, Inc.