Glycogen storage disease type Ia: molecular study in Brazilian patients

Mutations in the glucose-6-phosphatase (G6Pase) gene are responsible for glycogen storage disease type Ia (GSDIa). This disease is characterized by growth retardation, hepatomegaly, hypoglycemia, hyperlipidemia, and lactic acidosis. In this study, we report mutations in the G6Pase gene in 8 of 25 Brazilian patients with clinical symptoms of GSDIa. Five previously described mutations (R83C, Q347X, V338F, D38V, and G68R) were detected. The two most common mutations identified were R83C and Q347X, accounting for 8 of 14 (57.14%) mutant alleles. A 1176 single-nucleotide polymorphism and two intronic mutations (IVS3-58T>A and IVS4+10G>A) were also analyzed. We used the minigene strategy in order to verify the effect of these intronic mutations on the splicing mechanism. This study emphasizes that molecular genetic analysis is a reliable and convenient alternative to the assay of enzyme activity in a fresh liver biopsy specimen for diagnosing GSDIa. Received: November 13, 2000 / Accepted: December 25, 2000     

Canavan disease: carrier-frequency determination in the Ashkenazi Jewish population and development of a novel molecular diagnostic assay

Canavan disease (CD) is an autosomal recessive progressive neurodegenerative disorder prevalent in the Ashkenazi Jewish (AJ) population. The carrier rate for the most common mutations that cause CD in the AJ population is often quoted as 1:37-1:40. This is not supported by our finding of only two diagnosed cases of CD in the last 20 years in the Toronto AJ population of 160,000 and an estimated birth rate of 1,500-2,000 per year. Therefore, we embarked on a prevalence cross-sectional screening study to determine the carrier rate of CD in this population. In order to perform low-cost, high-throughput population testing for CD using molecular techniques, we first developed a novel molecular assay using multiplex fluorescent allele specific polymerase chain reaction (PCR) to test for the three most common mutations causing CD in the AJ population (A854C, C693A, C914A) and a neutral polymorphism at the site of the C693A mutation. During testing it was noted that individuals who were carriers of the A854C mutation also had a T polymorphism at the site of the C693A mutation (Y231X). We confirmed that in all A854C carriers the 854C mutation was in disequilibrium with the 693T polymorphism, indicating a founder chromosome for the A854C mutation in the AJ population. Twenty-five carriers were found from 1,423 samples yielding a carrier rate of 1:57, differing from the widely quoted frequency of 1:40 and supporting our observed frequency of disease.     

The frequency of mucolipidosis type IV in the Ashkenazi Jewish population and the identification of 3 novel MCOLN1 mutations

Mucolipidosis type IV (MLIV) is a neurodegenerative lysosomal storage disorder that occurs in an increased frequency in the Ashkenazi Jewish (AJ) population. The frequency of the disease in this population has been established by the testing of 66,749 AJ subjects in the Dor Yeshorim program, a unique premarital population-screening program designed for the Orthodox Jewish community. A carrier rate of 0.0104 (95% C.I 0.0097-0.011) was found. The distribution of the 2 AJ founder mutations, namely, c.416-2A>G and c.1_788del, was determined to be 78.15% and 21.85%, respectively. Three novel mutations were identified in non-Jewish MLIV patients, a missense mutation c.1207C>T, p.Arg403Cys; a 2bp deletion, c.302_303delTC; and a nonsense, c.235C>T, Gln79X.     

Familial dysautonomia: detection of the IKBKAP IVS20(+6T --> C) and R696P mutations and frequencies among Ashkenazi Jews

Familial dysautonomia (FD) is an autosomal recessive congenital neuropathy that occurs almost exclusively in the Ashkenazi Jewish (AJ) population. Mutations in the IkappaB kinase complex-associated protein (IKBKAP) gene cause FD. Two IKBKAP mutations, IVS20(+6T --> C) and R696P, have been identified in FD patients of AJ descent. The splice site mutation IVS20(+6T --> C) is responsible for > 99.5% of known AJ patients with FD, and haplotype analyses were consistent with a common founder. In contrast, the R696P mutation has been identified in only a few AJ patients. To facilitate carrier detection, a single PCR and allele-specific oligonucleotide (ASO) hybridization assay was developed to facilitate the detection of both the IVS20(+6T --> C) and R696P mutations. Screening of 2,518 anonymous AJ individuals from the New York metropolitan area revealed a carrier frequency for IVS20(+6T --> C) of 1 in 32 (3.2%; 95% CI, 2.5-3.9%), similar to the previously estimated carrier frequency (3.3%) based on disease incidence. No carrier was identified for the R696P lesion, indicating that the mutation was rare in this population (< 1 in 2,500). This sensitive and specific assay should facilitate carrier screening for FD mutations in the AJ community, as well as postnatal diagnostic testing.     

Premarital and prenatal screening for cystic fibrosis: experience in the Ashkenazi Jewish population.

PURPOSE: Since the early 1990s, Dor Yeshorim (DY) and the Mount Sinai School of Medicine (MSSM) have conducted premarital and prenatal carrier screening for cystic fibrosis (CF) in the Ashkenazi Jewish (AJ) population as part of their genetic testing programs, respectively. Together, over 170,000 screenees have been tested. In this study, we report the CF mutation frequencies in over 110,000 screenees who reportedly were of 100% AJ descent from the DY program and MSSM. In addition, the CF mutation frequencies in a group of > 7,000 screenees for AJ diseases who were of < 100% AJ descent are reported. METHODS: Testing for CF mutations was performed by either PCR and restriction digestion or ASO hybridization analyses at MSSM or sent to various academic and commercial laboratories by DY. RESULTS: The overall (and individual) carrier frequency for the five common AJ mutations, W1282X (0.020), DeltaF508 (0.012), G542X (0.0024), 3849+10kb C>T (0.0020), and N1303K (0.0016), among screenees who were 100% AJ was 1 in 26; when D1152H and the rare 1717-1G>A were included, the overall carrier frequency increased to approximately 1 in 23. In four families with D1152H, five compound heterozygotes for D1152H and W1282X (n = 2), DeltaF508 (1) or 3849+10kb C>T (1) were identified. In contrast, the carrier frequency for screenees reporting < 100% AJ descent was approximately 1 in 30 for the seven mutations. CONCLUSIONS: The carrier frequency for five common CF mutations in a large 100% AJ sample increased from 1 in 26 to 1 in 23 when D1152H was included in the panel. Addition of D1152H to mutation panels when screening the AJ population should be considered because compound heterozygosity is associated with a variable disease phenotype. Further studies to delineate the phenotype of CF patients with this mutation are needed.     

Prepregnancy body mass index and risk of multiple congenital anomalies

Familial dysautonomia (FD) is an autosomal recessive congenital neuropathy that occurs almost exclusively in the Ashkenazi Jewish (AJ) population. Mutations in the IκB kinase complex‐associated protein (IKBKAP) gene cause FD. Two IKBKAP mutations, IVS20^{+6T → C} and R696P, have been identified in FD patients of AJ descent. The splice site mutation IVS20^{+6T → C} is responsible for > 99.5% of known AJ patients with FD, and haplotype analyses were consistent with a common founder. In contrast, the R696P mutation has been identified in only a few AJ patients. To facilitate carrier detection, a single PCR and allele‐specific oligonucleotide (ASO) hybridization assay was developed to facilitate the detection of both the IVS20^{+6T → C} and R696P mutations. Screening of 2,518 anonymous AJ individuals from the New York metropolitan area revealed a carrier frequency for IVS20^{+6T → C} of 1 in 32 (3.2%; 95% CI, 2.5–3.9%), similar to the previously estimated carrier frequency (3.3%) based on disease incidence. No carrier was identified for the R696P lesion, indicating that the mutation was rare in this population (< 1 in 2,500). This sensitive and specific assay should facilitate carrier screening for FD mutations in the AJ community, as well as postnatal diagnostic testing. © 2002 Wiley‐Liss, Inc.     

The GALT rush: high carrier frequency of an unusual deletion mutation of the GALT gene in the Ashkenazi population

Classic galactosemia is an autosomal recessive disorder of galactose metabolism manifesting in the first weeks of life following exposure to a milk-based diet. Despite the benefit of avoidance of lactose, many patients suffer from long-term complications including neurological deficits and ovarian failure. To date, over 230 mutations have been described in the GALT gene resulting in galactosemia. Recently, an unusual mutation was characterized causing a 5.5 kb deletion, with a relatively high carrier rate in subjects of Ashkenazi Jewish (AJ) descent. The aim of this study was to estimate the carrier frequency of this mutation in the AJ population in Israel. For this purpose we developed a high-throughput methodology to genotype both normal and deleted alleles using a chip-based matrix-assisted laser desorption-time-of-flight (MALDI-TOF) mass spectrometer and Multiplex PCR. DNA samples of 760 anonymous AJ subjects were submitted for analysis, subsequently detecting six individuals heterozygous for the GALT deletion mutation, giving a carrier frequency of 1 in 127 (0.79%). Based on these results, we suggest that the method described here provides a basis for genetic screening and prenatal counseling and can potentially reduce the morbidity and mortality associated with delayed diagnosis of galactosemia in this patient population.     

Identification of mutations in the glucose-6-phosphatase gene in Czech and Slovak patients with glycogen storage disease type ia, including novel mutations K76N, V166A and 540del5

Mutations in the glucose-6-phosphatase (G6Pase) gene are responsible for glycogen storage disease type Ia (GSD Ia). A study of the molecular basis of GSD Ia was carried out in 12 Czech and Slovak GSD Ia patients from 10 unrelated families. Mutation analysis was performed for the entire coding region of G6Pase gene using DGGE, sequencing and PCR/digestion. With the strategy used, all mutant alleles were identified in this study. Three novel mutations (K76N, V166A and 540del5), six previously described mutations (W77R, R83C, G188R, R295C, Q347X and 158delC) and one known polymorphism (1176T-->C) were detected. The most common mutation identified was R83C, accounting for 8 out of 20 (40%) mutant alleles. The K76N mutation was found in a Gypsy family: two siblings with GSD Ia were homozygous for this mutation. These findings expand our knowledge of mutations responsible for glycogen storage disease type Ia.     

Mucolipidosis type IV: novel MCOLN1 mutations in Jewish and non-Jewish patients and the frequency of the disease in the Ashkenazi Jewish population

The gene MCOLN1 is mutated in Mucolipidosis type IV (MLIV), a neurodegenerative, recessive, lysosomal storage disorder. The disease is found in relatively high frequency among Ashkenazi Jews due to two founder mutations that comprise 95% of the MLIV alleles in this population [Bargal et al., 2000]. In this report we complete the mutation analysis of Jewish and non-Jewish MLIV patients whose DNA were available to us. Four novel mutations were identified in the MCOLN1 gene of severely affected patients: two missense, T232P and F465L; a nonsense, R322X; and an 11-bp insertion in exon 12. The nonsense mutation (R322X) was identified in two unrelated patients with different haplotypes in the MCOLN1 chromosomal region, indicating a mutation hotspot in this CpG site. An in-frame deletion (F408del) was identified in a patient with unusual mild psychomotor retardation. The frequency of MLIV in the general Jewish Ashkenazi population was estimated in a sample of 2,000 anonymous, unrelated individuals assayed for the two founder mutations. This analysis indicated a heterozygotes frequency of about 1/100. A preferred nucleotide numbering system for MCOLN1 mutations is presented and the issue of a screening program for the detection of high-risk families in the Jewish Ashkenazi population is discussed.     

Determination of cystic fibrosis carrier frequency for Zuni native Americans of New Mexico

The Zuni native Americans of the Southwest have an incidence of cystic fibrosis of approximately one in 333 or seven and one-half times that found for Caucasians. Earlier studies indicated that dF508 was not among the cystic fibrosis mutations causing this disease. Through a collaborative study the R1162X mutation was found on 12 out of 12 cystic fibrosis chromosomes from six Zuni patients. Because of the relative high incidence of cystic fibrosis, we undertook a study to determine the carrier frequency of the R1162X mutation among randomly sampled individuals. We found the carrier frequency in the general population for the R1162X to be 6.7%, a very significant number when compared with the carrier frequency for all cystic fibrosis mutations in the Caucasian population of approximately 4%.     

E148Q MEFV mutation carriage and longevity in individuals of Ashkenazi origin

The high carriage rate of MEFV mutations in at risk populations suggests that they confer a selective advantage, possibly by way of protection from infections. Here, we sought to assess whether this putative protection contributes to longevity, by studying MEFV mutation status in nonagenarians and the association of mutation carriage with life-threatening conditions. DNA samples and a medical history questionnaire were obtained from 200 nonagenarians (>90 years of age), who received medical treatment at a large tertiary hospital in Israel. The prevalence of MEFV mutations in the study group was compared to the known prevalence, by ethnic group, in the Israeli population. The presence of associated diseases in mutation carriers versus noncarriers was compared. The majority of study subjects were females (67.5 %) of Ashkenazi origin (78 %). A fifth carried an MEFV mutation, most commonly E148Q (73 % of total mutations), followed by V726A (5 %). Only the frequency of E148Q in Ashkenazi subjects was found to be higher than expected in the general Ashkenazi population (19.8 vs. 2.6 %, p < 0.0001). Cardiac arrhythmias and hypothyroidism were more common in mutation carriers, while no difference was noted, between carriers and noncarriers, in the rates of ischemic heart disease, diabetes, stroke and a wide range of other serious conditions. Our findings suggest that E148Q carriage contributes to longevity in the Ashkenazi population, perhaps by enhancing resistance to infections.     

Mutation analysis in 24 French patients with glycogen storage disease type 1a.

Both alleles of 24 French glycogen storage disease type 1a patients were sequenced: 14 different mutations allowed the identification of complete genotypes for all the patients. Nine new gene alterations are reported. Five mutations, Q347X, R83C, D38V, G188R, and 158 del C, account for 75% of the mutated alleles. These data show that the molecular pathology of the glucose-6-phosphatase gene is heterogeneous in this population. Complete genotyping of the index case by systematic sequencing is necessary to allow prenatal diagnosis in chorionic villi for at risk couples.     

Prevalence of glucocerebrosidase mutations in the Israeli Ashkenazi Jewish population

Gaucher disease is the most prevalent inherited disease among Ashkenazi Jews. It is very heterogeneous due to a large number of mutations within the glucocerebrosidase gene, whose impaired activity is the cause for this disease. Aiming at determining Gaucher carrier frequency among the Ashkenazi Jewish population in Israel, 1,208 individuals were molecularly diagnosed for six mutations known to occur among Ashkenazi Jewish Gaucher patients, using the newly developed Pronto™ Gaucher kit. The following mutations were tested: N370S, 84GG, IVS2+1, D409H, L444P, and V394L. Molecular testing of these mutations also allows identification of the recTL allele. The results indicated that Gaucher carrier frequency is 1:17 within the tested population. The prevalence of N370S carriers is 1:17.5. This implies that ˜1:1225 Ashkenazi Jews will be homozygous for the N370S mutation. Actually, in our study of 1,208 individuals one was found to be homozygous for the N370S mutation. The actual number of known Ashkenazi Jewish Gaucher patients with this genotype is much lower than that expected according to the frequency of the N370S mutation, suggesting a low penetrance of this mutation. Results of loading experiments in cells homozygous for the N370S mutation, as well as cells homozygous for the L444P and the D409H mutations, exemplified this phenomenon. Hum Mutat 12:240–244, 1998. © 1998 Wiley-Liss, Inc.     

Frequency of the Tay-Sachs disease splice and insertion mutations in the UK Ashkenazi Jewish population.

Tay-Sachs disease is a lethal neurodegenerative disorder caused by deficiency of the lysosomal enzyme beta-hexosaminidase A and inherited in an autosomal recessive fashion; carriers of the disease are 10 times more frequent in the Ashkenazi Jewish community than in the general population. Over 90% of North American Ashkenazi carriers tested have been shown to have either a splice site mutation at the boundary of exon 12 and intron 12 in the beta-hexosaminidase alpha subunit gene, or a 4 base pair insertion in exon 11. We describe simple assays involving amplification of DNA across these two mutation sites by polymerase chain reaction and the results of screening 75 subjects are given. The frequencies of the splice and insert mutations in 41 UK Ashkenazi carriers (20% to 80%, respectively) were similar to those found in the North American community. Twelve Ashkenazi subjects classified as non-carriers on enzyme assay were found to be negative for both mutations tested. All Ashkenazi carriers tested (both obligate carriers and those picked up by population screening) had either the splice or insert mutations; in contrast to this, only 21% of the non-Ashkenazi carriers had one or other of these mutations. It is concluded that within the carrier screening programmes for the Ashkenazi community, assays for the splice and insert mutations, together with an assay recently described for a mutation causing the rarer adult onset form of the disease, will prove useful as confirmatory tests for subjects who give positive or borderline results when screened on enzyme assay.     

A novel mutation in the HEXA gene specific to Tay-Sachs disease carriers of Jewish Iraqi origin

An increased frequency of carriers of 1:140, as defined by reduced hexosaminidase A (HexA) activity, was observed among Iraqi Jews participating in the Tay-Sachs disease (TSD) carrier detection program. Prior to this finding, TSD among Jews had been restricted to those of Eastern European (Ashkenazi) and Moroccan descent with carrier frequencies of 1:29 and 1:110 for Jews of Ashkenazi and Moroccan extraction, respectively. A general, pan-ethnic frequency of approximately 1:280 has been observed among other Jewish Israeli populations. Analysis of 48 DNA samples from Iraqi Jews suspected, by enzymatic assay, to be carriers revealed a total of five mutations, one of which was novel. In nine carriers (19%), a known mutation typical to either Ashkenazi or Moroccan Jews was identified. DeltaF304/ 305 was detected in four individuals, and + 1278TATC in three. G269S and R170Q each appeared in a single person. The new mutation, G749T, resulting in a substitution of glycine to valine at position 250 has been found in 19 of the DNA samples (40%). This mutation was not detected among 100 non-carrier, Iraqi Jews and 65 Ashkenazi enzymatically determined carriers. Aside from Ashkenazi and Moroccan Jews, a specific mutation in the HEXA gene has now also been identified in Jews of Iraqi descent.     

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.     

Identification of the first non-Jewish mutation in familial Dysautonomia

Familial Dysautonomia is an autosomal recessive disease with a remarkably high carrier frequency in the Ashkenazi Jewish population. It has recently been estimated that as many as 1 in 27 Ashkenazi Jews is a carrier of FD. The FD gene has been identified as IKBKAP, and two disease-causing mutations have been identified. The most common mutation, which is present on 99.5% of all FD chromosomes, is an intronic splice site mutation that results in tissue-specific skipping of exon 20. The second mutation, R696P, is a missense mutation that has been identified in 4 unrelated patients heterozygous for the major splice mutation. Interestingly, despite the fact that FD is a recessive disease, normal mRNA and protein are expressed in patient cells. To date, the diagnosis of FD has been limited to individuals of Ashkenazi Jewish descent and identification of the gene has led to widespread diagnostic and carrier testing in this population. In this report, we describe the first non-Jewish IKBKAP mutation, a proline to leucine missense mutation in exon 26, P914L. This mutation is of particular significance because it was identified in a patient who lacks one of the cardinal diagnostic criteria for the disease-pure Ashkenazi Jewish ancestry. In light of this fact, the diagnostic criteria for FD must be expanded. Furthermore, in order to ensure carrier identification in all ethnicities, this mutation must now be considered when screening for FD.     

Experience with carrier screening and prenatal diagnosis for 16 Ashkenazi Jewish genetic diseases

The success of prenatal carrier screening as a disease prevention strategy in the Ashkenazi Jewish (AJ) population has driven the expansion of screening panels as disease‐causing founder mutations have been identified. However, the carrier frequencies of many of these mutations have not been reported in large AJ cohorts. We determined the carrier frequencies of over 100 mutations for 16 recessive disorders in the New York metropolitan area AJ population. Among the 100% AJ‐descended individuals, screening for 16 disorders resulted in ～1 in 3.3 being a carrier for one disease and ～1 in 24 for two diseases. The carrier frequencies ranged from 0.066 (1 in 15.2; Gaucher disease) to 0.006 (1 in 168; nemaline myopathy), which averaged ～15% higher than those for all screenees. Importantly, over 95% of screenees chose to be screened for all possible AJ diseases, including disorders with lower carrier frequencies and/or detectability. Carrier screening also identified rare individuals homozygous for disease‐causing mutations who had previously unrecognized clinical manifestations. Additionally, prenatal testing results and experience for all 16 disorders (n = 574) are reported. Together, these data indicate the general acceptance, carrier frequencies, and prenatal testing results for an expanded panel of 16 diseases in the AJ population. Hum Mutat 31:1–11, 2010. © 2010 Wiley‐Liss, Inc.     

CFTR gene: molecular analysis in patients from South Brazil

Cystic fibrosis (CF) is the most common genetic disease among Caucasians. The CF gene, named cystic fibrosis transmembrane conductance regulator (CFTR), codifies a protein that acts as a channel through the epithelial membrane. The present work aimed (1) to detect sequence alterations in the nucleotide binding regions and at the membrane spanning domain of the CFTR gene and (2) to detect the following frequent mutations R347P, R347H, R334W, and Q359K (located in exon 7), DeltaF508 (located in exon 10), G542X, G551D, R553X, and S549N (located in exon 11), W1282X (located in exon 20), and N1303K (located in exon 21). Seventy-seven unrelated CF patients were analyzed, who were previously diagnosed and currently under treatment at the Pneumology Service of our hospital. Regions of interest were amplified by PCR using specific primers. Each sample was analyzed by a non-radioactive single-stranded conformational polymorphism (SSCP) analysis technique and restriction enzyme digestion. The DeltaF508 mutation was found in 48.7% of the alleles. Frequencies of G542X, R334W, R553X, and W1282X mutations in our population were 3.25, 1.3, 0.65, and 0.65%, respectively. No alleles were found to carry mutations G551D, R334W, R347P, R347H, Q359K, S549N, and N1303K, which were included in the screening protocol. This study allowed the characterization of 84 out of 154 CF mutant alleles (54.5%). The incidence of main CF mutations analyzed was similar to that of the south European population. Mutation data presented here will be useful for designing new DNA testing strategies for CF in South Brazil.     

Carrier frequency of two BBS2 mutations in the Ashkenazi population

Bardet–Biedl syndrome (BBS) is known to be caused by numerous mutations that occur in at least 15 of the BBS genes. As the disease follows an autosomal recessive pattern of inheritance, carrier screening can be performed for at‐risk couples, but the number of potential mutation sites to screen can be daunting. Ethnic studies can help to narrow this range by highlighting mutations that are present at higher percentages in certain populations. In this article, the carrier frequency for two mutations that occur in the BBS2 gene, c.311A>C and c.1895G>C were studied in individuals of Ashkenazi Jewish descent in order to advise on including them in existing mutation panels for this population. Carrier screenings were performed on individuals from the Ashkenazi Jewish population using a combination of TaqMan genotyping assays followed by real‐time polymerase chain reaction (PCR) and allelic discrimination, and allele‐specific PCR confirmed by restriction analysis. The combined results indicated carrier frequencies of 0.473% (±0.0071%) for the c.311A>C mutation and 0.261% (±0.0064%) for the c.1895G>C mutation. On the basis of these frequencies, we believe that the two mutations should be considered for inclusion in screening panels for the Ashkenazi population.