Ethnic effect on FMR1 carrier rate and AGG repeat interruptions among Ashkenazi women.

PURPOSE: Fragile X syndrome, a common cause of intellectual disability, is usually caused by CGG trinucleotide expansion in the FMR1 gene. CGG repeat size correlates with expansion risk. Premutation alleles (55-200 repeats) may expand to full mutations in female meiosis. Interspersed AGG repeats decrease allele instability and expansion risk. The carrier rate and stability of FMR1 alleles were evaluated in large cohorts of Ashkenazi and non-Ashkenazi women. METHODS: A total of 4,344 Ashkenazi and 4,985 non-Ashkenazi cases were analyzed using Southern blotting and polymerase chain reaction between 2004 and 2011. In addition, AGG interruptions were evaluated in 326 Ashkenazi and 298 non-Ashkenazi women who were recruited during 2011. RESULTS: Both groups had major peaks of 30 and 29 repeats. Ashkenazi women had a higher frequency of 30 repeats and a lower frequency of other peaks (P < 0.0001). A higher rate of premutations in the 55-59 repeats range (1:114 vs. 1:277) was detected among the Ashkenazi women. Loss of AGG interruptions (<2) was significantly less common among Ashkenazi women (9 vs. 19.5% for non-Ashkenazi women, P = 0.0002). CONCLUSION: Ashkenazi women have a high fragile X syndrome carrier rate and mostly lower-range premutations, and carry a low risk for expansion to a full mutation. Normal-sized alleles in Ashkenazi women have higher average number of AGG interruptions that may increase stability. These factors may decrease the risk for fragile X syndrome offspring among Ashkenazi women.

A compound heterozygous missense mutation and a large deletion in the KCTD7 gene presenting as an opsoclonus-myoclonus ataxia-like syndrome.

Mutations in the potassium channel-related gene KCTD7 were described so far in a single family with progressive myoclonus epilepsy. We describe a unique phenotype: acute onset of myoclonus and ataxia, associated with abnormal opsoclonus-like eye movements; improvement of clinical symptoms under steroid treatment; and appearance of epileptic activity on EEG 2 years later without overt seizures. After excluding possible genetic causes, whole-genome exome sequencing was performed in order to identify the causative gene. One heterozygous missense mutation (R84W) was detected by exome sequencing and a large heterozygous deletion of exons 3 and 4 by MLPA analysis. The father is heterozygous for the R84W mutation and the mother is heterozygous for the exon 3+4 deletion. The mutation affects a highly conserved segment of the predicted protein, changing a basic amino acid into neutral. The large deletion probably results in a truncated protein. The different phenotype broadens the spectrum of KCTD7-related diseases. Therefore, patients diagnosed as having opsoclonus-myoclonus with an atypical course should be evaluated for KCTD7 mutations.

Predictive value of TP53 fluorescence in situ hybridization in cytogenetic subgroups of acute myeloid leukemia.

Acute myeloid leukemia (AML) with a complex karyotype (CK) has frequent alterations in TP53 and a very poor prognosis. We examined whether a prompt and simple fluorescence in situ hybridization (FISH) analysis for 17p13 deletion at diagnosis has a predictive value for response to therapy and overall survival in subgroups of AML. In 15 patients with a normal karyotype the TP53 FISH analysis was normal, whereas in 16 patients with CK 75% had only one copy of the TP53 allele. The deletion was also detected in 33% of six patients with monosomy or partial monosomy of chromosome 5, 7, 9, or 12. This loss of TP53 correlated significantly with a poor response to chemotherapy, and the median survival time of these patients was shorter. TP53 FISH analysis carried out at diagnosis has a predictive value with respect to chemotherapy response and can therefore facilitate a rapid decision on treatment strategies.

Large-scale population screening for spinal muscular atrophy: clinical implications.

PURPOSE: To determine the frequency of SMN1 deletion carriers in the Israeli population and to assess the feasibility of population screening for spinal muscular atrophy. METHODS: A total of 6394 individuals without family history of spinal muscular atrophy underwent genetic screening by multiplex ligation-dependent probe amplification, designed to detect SMN1 exon 7 and exon 8 copy number. RESULTS: One hundred fifty-nine individuals carried an SMN1 heterozygous exon 7 deletion, yielding a carrier frequency of 1:40. About 10.8% of individuals were found to carry two or more SMN1 exon 7 copies on the same chromosome (cis configuration). This implies that some deletion carriers may not be detected by multiplex ligation-dependent probe amplification or similar quantitative methods. The acceptance of spinal muscular atrophy screening among women undergoing testing for fragile X syndrome and cystic fibrosis reached 93%. CONCLUSIONS: Currently used molecular techniques cannot detect about 5% of spinal muscular atrophy carriers with a cis configuration or individuals with SMN1 sequence mutations and de novo deletions. Thus, it is estimated that the spinal muscular atrophy carrier detection rate is about 90%. Given the severity of spinal muscular atrophy, the relatively high carrier frequency, and the estimated detection rate, we conclude that population-based screening for spinal muscular atrophy is feasible and acceptable.

Clinical and genetic heterogeneity of congenital adrenal hypoplasia due to NR0B1 gene mutations.

INTRODUCTION: X-linked adrenal hypoplasia congenita (AHC) is a rare disorder caused by mutations or complete deletion of the NR0B1 gene that encodes the DAX-1 protein, an orphan member of the nuclear receptor superfamily. AHC is characterized by adrenal insufficiency in infancy and early childhood. Later, hypogonadotropic hypogonadism (HH) manifests as pubertal failure. PATIENTS AND METHODS: We evaluated the clinical, endocrine and molecular characteristics of 12 AHC patients from 5 families diagnosed between 1984 and 2007 in Israel. RESULTS: Most of the boys (10/12) presented with signs of adrenal insufficiency such as salt wasting and failure to thrive during the neonatal period. Aldosterone deficiency usually preceded cortisol deficiency requiring early mineralocorticoid therapy. Serum cortisol levels in the first weeks of life varied from very low to high levels (<2.76 to >1776 nmol/l). Five boys showed signs of precocious sexual development during infancy and childhood, including enlargement of the penis and testes. In four patients the initial diagnoses were erroneous. Molecular analysis of the NR0B1 gene identified point mutations in six patients including a novel splice site mutation in one patient and his family (IVS1-1G-->C). Contiguous gene deletion was found in six patients from two families who manifested impaired mental development. CONCLUSIONS: In X-linked AHC caused by different molecular defects in NR0B1 gene, the clinical spectrum of the disease is quite variable and precocious sexual development is a prominent feature. Genetic testing is indicated in boys presenting with salt-wasting with or without cortisol deficiency if congenital adrenal hyperplasia has been ruled out.

Cytogenetic analysis of 101 skull base tumors.

BACKGROUND: Skull base tumors are rare neoplasms and the cytogenetic data on these tumors are limited. The authors cytogenetically analyzed a large series of tumors and compared the findings with patients' pathologic data. METHODS: The karyotypes of pathologically confirmed samples of 101 patients, who were operated for oncological extirpation of tumors, were analyzed using G-banding and spectral-karyotyping techniques. RESULTS: Of the 67 malignant tumors, 32 (48%) had chromosomal aberrations, some with complex numerical and structural chromosomal anomalies. Recurrent chromosomal breakpoints were identified in squamous cell carcinomas, adenoid cystic carcinomas (ACCs), sinonasal undifferentiated carcinomas, chordomas, and sarcomas. Specific breakpoints established the diagnosis of various soft tissue sarcomas. Novel chromosomal aberrations were found in various other malignant and benign tumors. CONCLUSION: This study highlights the value of cytogenetic analysis for diagnosis of skull base tumors. The data add further information on the biological behavior of these rare neoplasms.

Cytogenetic analysis of sinonasal carcinomas.

BACKGROUND: Sinonasal carcinomas, including nonkeratinizing (NK) squamous cell carcinoma (SCC) and sinonasal undifferentiated carcinoma (SNUC), are uncommon malignant neoplasms arising from the Schneiderian respiratory epithelium of the nasal cavity and paranasal sinuses. Due to their low frequency, the cytogenetic data on these tumors is limited. METHODS: Seventeen patients who were operated on in our institution for extirpation of paranasal carcinomas were enrolled in this study. Fourteen pathologically confirmed samples of sinonasal carcinomas were cytogenetically analyzed using G-banding techniques after short-term culture. Three samples did not grow on culture. RESULTS: Five of the 14 sinonasal carcinomas had an abnormal karyotype (36%). Of the 9 NK SCCs, 3 had abnormal karyotypes with numerical and structural chromosomal anomalies. Of the 5 patients with SNUC, 2 had an abnormal karyotype. One case of SNUC had a diploid complex karyotype. Another case of SNUC had a near triploid composite karyotype with 60-69 chromosomes. The chromosome arms that involved frequent breakpoint and rearrangements were: 1p, 6p, 7p, and 12q. We found that 3 of the 3 patients who died of disease displayed an abnormal karyotype, whereas 2 of the 11 patients who are alive displayed an abnormal karyotype (P = 0.027). CONCLUSIONS: The study revealed that more than a third of the paranasal carcinomas carry an abnormal karyotype. No specific common aberrations were found in these tumors. To our knowledge this is the first attempt to investigate sinonasal squamous and undifferentiated carcinomas on a genetic level using G-banding technique. Additional studies are required in order to determine whether cytogenetic data may serve as an adjunct to conventional pathology for the diagnosis and prognosis assessment of these rare and highly aggressive tumors.

Multiplex nested PCR for preimplantation genetic diagnosis of spinal muscular atrophy.

OBJECTIVE: Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder caused in most patients by homozygous deletion of the SMN1 gene. For a carrier couple at a 25% risk of affected offspring, preimplantation genetic diagnosis (PGD) offers an alternative to prenatal diagnosis and termination of affected pregnancies. Our objective was to develop an accurate and reliable single-cell multiplex nested PCR analysis for PGD of SMA. METHODS: The method was developed on single blood leukocytes, obtained from healthy controls and an adult SMA type III patient with a known homozygous deletion of SMN1 exon 7 and 8. Multiplex nested PCR on single cells was used to co-amplify exons 7 and 8 of SMN. Additional multiplexing was performed with the ZFX/ZFY gene for sexing. Following successful establishment of the multiplex nested PCR protocol in single leukocytes, the technique was employed for PGD in 4 patients for a total of 7 cycles. In 2 patients, sexing was simultaneously performed using ZFX/ZFY. RESULTS: 220 single leukocytes from a normal individual and 220 from an SMA patient were analyzed. Exon 7 of SMN1 was amplified in 99% of normal single leukocytes and in none of the SMA-affected leukocytes. Exon 7 of SMN2 was amplified in 100% of both normal and SMA-affected leukocytes. Exon 8 of SMN1 was amplified in 98% of normal cells and in none of the SMA-affected leukocytes. Exon 8 of SMN2 was amplified in 96% of both normal and SMA-affected leukocytes. Amplification efficiency was 99% for ZFX/ZFY. There were no false-negative results and no contamination was detected in all wash-drop blanks tested. Seven PGD cycles were performed in 4 SMA-carrier couples with successful molecular analysis of 34 embryos and a total of 15 normal embryos transferred in 7 cycles. One clinical pregnancy has resulted in the delivery of a healthy male. Amniocentesis performed at 17 weeks confirmed the correct diagnosis for both SMA and sexing. CONCLUSIONS: These results suggest that our multiplex nested PCR protocol offers an efficient and accurate method for PGD of SMA while enabling the simultaneous analysis of an additional loci.

Screening for familial dysautonomia in Israel: evidence for higher carrier rate among Polish Ashkenazi Jews.

Familial dysautonomia (FD) is an autosomal recessive disorder characterized by hereditary sensory and autonomic neuropathies. Although extremely rare in most populations, FD is common among Ashkenazi Jews (AJ), with a calculated carrier frequency of 1 in 30, based on disease prevalence. The gene for FD was recently identified as IKBKAP. One major mutation (IVS2 + 6T --> C) is responsible in >99.5% of cases among AJ. The purpose of this study was to determine the actual frequency of FD carriers in the AJ population in Israel and to determine whether carriers are more frequent among a subpopulation of AJ from Poland. The study group included 1267 Jews of Ashkenazi origin who were referred for routine DNA screening tests. These included 1100 individuals who were full AJ and 167 who were part AJ. None had a family history of FD. Mutation analysis for (IVS2 + 6T --> C) was performed by PCR amplification followed by restriction enzyme analysis. All positive cases were confirmed by DHPLC WAVE( trade mark ). Among the 1100 full AJ tested, 34 were found to be FD carriers (1:32). The incidence of mutation carriers was significantly higher in AJ of Polish descent (1:18) compared to AJ of non-Polish descent (1:99). Among the 167 individuals who were part AJ, there were 3 carriers (1:56). The incidence of FD among AJ, particularly those of Polish background, warrants population screening. Population screening may be performed by denaturing high-performance liquid chromatography.

MECP2 mutations in Israel: implications for molecular analysis, genetic counseling, and prenatal diagnosis in Rett syndrome.

This report describes molecular analysis of the MECP2 gene in 37 Israeli patients suspected of having Rett syndrome (RTT). The patients were from various Jewish ethnic groups and from Arabic origin. Of the 17 patients with classical RTT, bi-directional sequencing of the coding exons revealed MECP2 mutations in 14 patients. About 66% of the mutations were located in previously described hot-spots. One case presented a novel mutation (141insA). Mutation-negative patients were further analyzed by Southern blot, which detected a novel gross rearrangement in another classical case. Altogether, detection rate in classical cases was 88%. In a non-classical case, a novel missense mutation (1451G>C) was detected in an affected girl and in her normal father, suggesting that this is a non-pathogenic alteration. Another variant (1461 + 96insA) was detected in an affected girl and in her healthy mother and also in another affected girl and her healthy father, suggesting that this variant too, is non-pathogenic. No significant difference in mutation type was noted among the different ethnic groups. In one familial case, the same mutation was detected in two sibs but not in their mother, suggesting germ-line mosaicism. Our results suggest that mutation-negative cases should be further assessed for gross rearrangements and that molecular analysis of the parents is often required when previously undescribed sequence alterations are detected.

Clinical and screening implications of the I1307K adenomatous polyposis coli gene variant in Israeli Ashkenazi Jews with familial colorectal neoplasia. Evidence for a founder effect.

BACKGROUND: The authors previously found the I1307K adenomatous polyposis coli (APC) gene variant in 5% of Ashkenazi control participants, in 15.4% of those who had familial colorectal neoplasia, but also in 1.6% of non-Ashkenazi control participants. In this study, they evaluated its use in a screening program for familial colorectal neoplasia and examined for a founder effect. METHODS: Consecutive Ashkenazim with a personal and/or family history of colorectal neoplasia had the DNA test. Markers flanking the APC gene were examined in Ashkenazi and non-Ashkenazi I1307K carriers and noncarriers. RESULTS: Among 718 persons, I1307K occurred in 6.2% of Ashkenazi participants, in 1.5% of non-Ashkenazi control participants (P = 0.02), and in 10.7% of Ashkenazim with familial neoplasia (relative risk, 1.73 [not significant compared with controls]; 95% confidence interval, 0.7-3.2). Colorectal neoplasia was detected in carriers at a younger age (P < 0.05) without excess risk for multiple colorectal neoplasia or noncolorectal neoplasia. I1307K attributable risk for colorectal neoplasia was 0.5-0.6%. Compared with noncarriers, both Ashkenazi and non-Ashkenazi I1307K carriers had similar flanking polymorphic alleles (P < 0.01). CONCLUSIONS: I1307K is a low-penetrance genetic variant that indicates a 1.7 relative risk for neoplasia in carriers who have familial carcinoma, clinically equivalent to obtaining a family history of sporadic colorectal neoplasia and promoting early screening. I1307K is a founder genetic variant in Jews of different ethnic origin, mainly Ashkenazim, but it explains only partially their higher incidence of colorectal carcinoma.

Molecular analysis of the APC gene in 71 Israeli families: 17 novel mutations.

Familial adenomatous polyposis (FAP) is caused by germline mutations in the APC gene. This study included 71 Israeli families referred for molecular analysis of the APC gene. Analysis was performed by the protein truncation test (PTT) of exon 15, and if negative, by direct sequencing of exon 1 to 14. Mutations were found in 36 (50.7%) probands. Mutation detection rates depended on the pattern of referral, such that among the 40 probands referred from the Service for Hereditary Cancer the mutation detection rate was 70%, whereas among the 31 probands referred by other gastroenterologists detection rate was significantly lower (25.8%). Of the 36 mutations detected, 21 were within exon 15, 13 within exons 1 to 14 and 2 were newly-described splicing mutations in introns 9 and 14. A relatively high proportion of the mutations was detected in exon 9 (6/36), five of them newly described. Altogether, we describe here 17 new mutations. Within the two major ethnic groups in Israel, patients of Ashkenazi and non-Ashkenazi origin, there was no significant differences in the mutation detection rate or the distribution of mutations within the APC gene. No founder mutation was detected in any of these populations. Our data confirm that higher detection rates may be expected in patients referred by clinical services specializing in hereditary colon cancer. These results further underscore the importance of complete analysis of all exons and exon/intron boundaries, in order to achieve maximal detection rate in patients suspected of FAP.

Rett syndrome: clinical manifestations in males with MECP2 mutations.

Rett syndrome is a neurodevelopmental disorder characterized by cognitive and adaptive regression with autistic features, loss of acquired skills, and stereotypic hand movements that almost exclusively affects females. It is an X-linked dominant disorder, with presumed lethality in males. Nonetheless, there are a few descriptions of males suspected of having Rett syndrome. With the recent discovery that the MECP2 gene is responsible for most cases of Rett syndrome, it is possible to molecularly assess cases of affected males by direct sequencing analysis. We describe an Israeli family consisting of a female having classic Rett syndrome and a male sibling with severe neonatal encephalopathy. Molecular analysis revealed that both sister and brother have the same MECP2 gene mutation; however, their mother does not. This case, as well as other published studies of males with MECP2 mutations, reveals that the clinical manifestations in viable males vary from neonates with severe encephalopathy to adults with mental retardation and demonstrate genotype-phenotype correlations.