Widened clinical spectrum of the Q128P MECP2 mutation in Rett syndrome.

CASE REPORT: We describe a female patient with Arnold Chiari type I malformation, atypical Rett syndrome characterized by postnatal onset microcephaly, stereotypic hand movements, ataxia, severe developmental delay, intractable tonic-clonic seizures, and a MECP2 mutation with a unique set of clinical findings. Implementation of a ketogenic diet resulted in decreased seizure activity and an improvement in the patient's degree of social relatedness with her family members. DISCUSSION: An early diagnosis of Rett syndrome allows families to maximize utilization of existing treatment modalities and seek appropriate genetic counseling and prenatal diagnoses. This case also provides further evidence for the treatment benefit of ketogenic diets for seizures in patients with Rett syndrome.

Juvenile onset Huntington disease resulting from a very large maternal expansion.

We report a 5(1/2)-year-old girl with a maternal family history of Huntington disease (HD), who presented clinically with unbalanced gait, impaired speech, and increasing difficulty with fine motor control. Onset of symptoms began at the age of 3(1/2) years. The suspected diagnosis of juvenile HD, based upon her family history, was confirmed by DNA analysis. At age 7, the patient died secondary to complications of her underlying disorder. Juvenile-onset Huntington disease is uncommon, predominantly transmitted by fathers and is always associated with very large expansions of the CAG repeat. Interestingly, this patient inherited a large CAG size expansion from her mother, who herself had symptoms of HD at the age of 18. Molecular analysis revealed that the mother had 70 CAG repeats whereas our patient had approximately 130 CAG repeats. This is the largest reported CAG expansion from a maternal transmission that has been confirmed molecularly and it demonstrates that very large expansions can also occur through the maternal lineage.

Clinical phenotype of Brazilian families with spinocerebellar ataxia 10.

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant ataxia caused by an ATTCT repeat expansion in an intron of the SCA10 gene. SCA10 has been reported only in Mexican families, in which the disease showed a combination of cerebellar ataxia and epilepsy. The authors report 28 SCA10 patients from five new Brazilian families. All 28 patients showed cerebellar ataxia without epilepsy, suggesting that the phenotypic expression of the SCA10 mutation differs between Brazilian and Mexican families.

Prenatal diagnosis of a homologous Robertsonian translocation involving chromosome 15.

We report the prenatal diagnosis of a fetus with a de novo Robertsonian translocation: 45,XY,der(15;15)(q10;q10). Although Robertsonian translocations are common chromosomal rearrangements, those involving homologous chromosomes are infrequent. Since chromosome 15 is imprinted, uniparental disomy (UPD) is a concern when chromosomal rearrangements involving chromosome 15 are identified. In the present case, UPD studies showed normal biparental inheritance. In contrast to the fact that most homologous acrocentric rearrangements are isochromosomes, these results indicate postzygotic formation of a Robertsonian translocation between biparentally inherited chromosomes 15.

Angiotensinogen and endothelial nitric oxide synthase gene polymorphisms among Hispanic patients with preeclampsia.

OBJECTIVE: We sought to establish an association between preeclampsia and the methionine to threonine polymorphism at amino acid residue 235 (Met235Thr) in angiotensinogen in a Hispanic population. We looked for a relationship between this allele and the allele in the endothelial nitric oxide synthase gene (NOS3) that produces the A form (NOS3*A) with respect to preeclampsia. STUDY DESIGN: Clinical data were collected from 87 patients with preeclampsia and 53 control subjects. Patients and controls were genotyped for the angiotensinogen polymorphism allele (AGT*T) and the NOS3*A polymorphism. We then compared patients with preeclampsia and control subjects and investigated disease severity within the preeclampsia group as a function of genotype. RESULTS: The AGT*T allelic frequencies among patients with preeclampsia and control subjects were 0.72 and 0.70, respectively (P =.84). The blood pressure of patients with an AGT*T allele who also carried a NOS3*A allele was higher at earlier gestational ages (r = -0.052; P =.02). Analysis suggested that the systolic blood pressure differences were due to gestational age effects and the presence of a NOS3*A allele (P <.10). CONCLUSION: The AGT*T allele was not associated with the development of preeclampsia. Independently of the presence of an AGT*T allele, the NOS3*A allele was associated with a higher blood pressure at an earlier gestational age.

Diagnostic testing for Rett syndrome by DHPLC and direct sequencing analysis of the MECP2 gene: identification of several novel mutations and polymorphisms.

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder affecting 1/10,000-15,000 girls. The disease-causing gene was identified as MECP2 on chromosome Xq28, and mutations have been found in approximately 80% of patients diagnosed with RTT. Numerous mutations have been identified in de novo and rare familial cases, and they occur primarily in the methyl-CpG-binding and transcriptional-repression domains of MeCP2. Our first diagnostic strategy used bidirectional sequencing of the entire MECP2 coding region. Subsequently, we implemented a two-tiered strategy that used denaturing high-performance liquid chromatography (DHPLC) for initial screening of nucleotide variants, followed by confirmatory sequencing analysis. If a definite mutation was not identified, then the entire MECP2 coding region was sequenced, to reduce the risk of false negatives. Collectively, we tested 228 unrelated female patients with a diagnosis of possible (209) or classic (19) RTT and found MECP2 mutations in 83 (40%) of 209 and 16 (84%) of 19 of the patients, respectively. Thirty-two different mutations were identified (8 missense, 9 nonsense, 1 splice site, and 14 frameshifts), of which 12 are novel and 9 recurrent in unrelated patients. Seven unclassified variants and eight polymorphisms were detected in 228 probands. Interestingly, we found that T203M, previously reported as a missense mutation in an autistic patient, is actually a benign polymorphism, according to parental analysis performed in a second case identified in this study. These findings highlight the complexities of missense variant interpretation and emphasize the importance of parental DNA analysis for establishing an etiologic relation between a possible mutation and disease. Overall, we found a 98.8% concordance rate between DHPLC and sequence analyses. One mutation initially missed by the DHPLC screening was identified by sequencing. Modified conditions subsequently enabled its detection, underscoring the need for multiple optimized conditions for DHPLC analysis. We conclude that this two-tiered approach provides a sensitive, robust, and efficient strategy for RTT molecular diagnosis.

Ashkenazi Jewish population frequency of the Bloom syndrome gene 2281 delta 6ins7 mutation.

Bloom syndrome is an autosomal recessive disorder characterized clinically by small size, sun-sensitive facial erythema, and immunodeficiency, and cytogenetically by increased chromosome breakage and sister chromatid exchange. Genomic instability renders Bloom syndrome patients at elevated risk for multiple cancers. Bloom syndrome occurs most commonly in the Ashkenazi Jewish population due to an apparent founder effect. The BLM gene on chromosome 15q26.1 was identified to encode a RecQ DNA helicase. Multiple mutations were identified, with Ashkenazi Jewish Bloom syndrome patients almost exclusively homozygous for a complex frameshift mutation (6-bp deletion/7-bp insertion at BLM nucleotide 2,281). This molecular genetic study seeks to verify the Ashkenazi Jewish carrier frequency of the BLM 2281 delta 6ins7 allele using semiautomated allele-specific oligonucleotide (ASO) analysis. Anonymized DNA samples from 1,016 Ashkenazi Jewish individuals and 307 non-Jewish individuals were screened. Ten Ashkenazi heterozygote carriers for the 2281 delta 6ins7 mutation were identified, giving a carrier frequency estimate of 0.98%, or approximately 1 carrier out of 102 individuals in the Ashkenazi Jewish population. These results are consistent with previous estimates, and combining our findings with the published molecular data collectively yields an Ashkenazi Jewish carrier frequency of approximately 1 in 104. Given its high population frequency and detection rate among Ashkenazi Jewish patients, the blmAsh mutation constitutes an appropriate addition to screening panels for Ashkenazi Jewish disease testing.

Applied molecular genetic techniques for prenatal diagnosis.

Molecular laboratory techniques are increasingly important in the evaluation of fetuses at risk for a single gene disorder or chromosomal abnormality and for the detection of genetic or other conditions that can lead to an adverse fetal or maternal outcome. The localization and identification of novel disease genes allows for mutation analysis or linkage studies on fetuses at risk for these disorders. New assays or techniques for mutation detection in single gene disorders such as amplification refractory mutation system polymerase chain reaction, fluorescent polymerase chain reaction, heteroduplex analysis and the protein truncation test are now applied in prenatal diagnosis. Recent advances in molecular cytogenetics, such as comparative genomic hybridization, the primed in-situ labeling technique, the development of new telomeric probes and spectral karyotyping, are being evaluated for their role in the prenatal diagnosis of chromosomal abnormalities. These methods may greatly improve the accuracy and applicability of preimplantation genetic diagnosis or diagnosis on fetal cells isolated from maternal blood.

Screening for 185delAG in the Ashkenazim.

A study was initiated to assess interest, educational effectiveness, and implications of genetic testing for the common BRCA1 mutation, 185delAG, in the Ashkenazim. Of 333 individuals who attended group sessions, 309 (92%) participated in the study. Participants were categorized as having negative family history (67%), positive family history (defined, by a relaxed criterion, as one first-degree relative or two second-degree relatives with breast [premenopausal] or ovarian cancer) (22%), positive personal history (7%), and both positive personal history and positive family history (4%). Group education was effective, as shown by the improvement in participant scores from pre- to posteducation tests. For the 289 individuals (94%) who requested testing, the major reasons included concern for their own risk, concern for the risk of their children, and desire to learn about surveillance options. The most common reason given by participants who declined testing was concern about health insurance. Six participants found to be heterozygous for the 185delAG mutation received results and were offered genetic counseling. Participants had consented for additional testing without receiving results and were screened for the 6174delT mutation in BRCA2, and seven were found to be positive. All identified carriers reported at least one first- or second-degree relative with a history of breast or ovarian cancer, although they did not all meet our study criteria for positive family history. Given these outcomes, we conclude that screening for breast and ovarian cancer susceptibility is most appropriate for individuals with a positive personal or positive family cancer history. We propose a guideline for future studies designed to identify individuals who may benefit from genetic testing for inherited breast and ovarian cancer.

Ashkenazi Jewish population frequencies for common mutations in BRCA1 and BRCA2.

BRCA1 and BRCA2 are the two major identified causes of inherited breast cancer, with mutations in either gene conferring up to 80-90% lifetime risk of breast cancer in carrier females. Mutations in BRCA1 account for approximately 45% of familial breast cancer and 90% of inherited breast/ovarian cancer, whereas mutations in BRCA2 account for a comparable percentage of inherited breast cancer cases. Over 85 distinct BRCA1 mutations and a growing list of BRCA2 mutations have been identified, with the majority resulting in protein truncation. A specific BRCA1 mutation, 185delAG, has a reported increased carrier frequency of approximately 0.9% in the Ashkenazi Jewish population, but is also found in rare non-Jewish patients with a different haplotype. The 6174delT mutation in BRCA2 was recently identified as a frequent mutation in 8 out of 107 Ashkenazi Jewish women diagnosed with breast cancer by age 50 (ref. 8), as well as in three Ashkenazi male breast cancer patients. We have conducted a large-scale population study to investigate the prevalence of specific BRCA1 and BRCA2 mutations in Ashkenazi Jewish individuals who were unselected for breast cancer. BRCA1 mutation screening on approximately 3,000 Ashkenazi Jewish samples determined a carrier frequency of 1.09% for the 185delAG mutation and 0.13% for the 5382insC mutation. BRCA2 analysis on 3,085 individuals from the same population showed a carrier frequency of 1.52% for the 6174delT mutation. This expanded population-based study confirms that the BRCA1 185delAG mutation and the BRCA2 6174delT mutation constitute the two most frequent mutation alleles predisposing to hereditary breast cancer among the Ashkenazim, and suggests a relatively lower penetrance for the 6174delT mutation in BRCA2.

Duplication of the PMP22 gene in 17p partial trisomy patients with Charcot-Marie-Tooth type-1 neuropathy.

Autosomal dominant Charcot-Marie-Tooth type-1A neuropathy (CMT1A) is a demyelinating peripheral nerve disorder that is commonly associated with a submicroscopic tandem DNA duplication of a 1.5-Mb region of 17p11.2p12 that contains the peripheral myelin gene PMP22. Clinical features of CMT1A include progressive distal muscle atrophy and weakness, foot and hand deformities, gait abnormalities, absent reflexes, and the completely penetrant electrophysiologic phenotype of symmetric reductions in motor nerve conduction velocities (NCVs). Molecular and fluorescense in situ hybridization (FISH) analyses were performed to determine the duplication status of the PMP22 gene in four patients with rare cytogenetic duplications of 17p. Neuropathologic features of CMT1A were seen in two of these four patients, in addition to the complex phenotype asociated with 17p partial trisomy. Our findings show that the CMT1A phenotype of reduced NCV is specifically associated with PMP22 gene duplications, thus providing further support for the PMP22 gene dosage mechanism for CMT1A.

Myelin protein zero (MPZ) gene mutations in nonduplication type 1 Charcot-Marie-Tooth disease.

The myelin protein zero gene (MPZ) maps to chromosome 1q22-q23 and encodes the most abundant peripheral nerve myelin protein. The Po protein functions as a homophilic adhesion molecule in myelin compaction. Mutations in the MPZ gene are associated with the demyelinating peripheral neuropathies Charcot-Marie-Tooth disease type 1B (CMT1B), and the more severe Dejerine-Sottas syndrome (DSS). We have surveyed a cohort of 70 unrelated patients with demyelinating polyneuropathy for additional mutations in the MPZ gene. The 1.5-Mb DNA duplication on chromosome 17p11.2-p12 associated with CMT type 1A (CMT1A) was not present. By DNA heteroduplex analysis, four base mismatches were detected in three exons of MPZ. Nucleotide sequence analysis identified a de novo mutation in MPZ exon 3 that predicts an Ile(135)Thr substitution in a family with clinically severe early-onset CMT1, and an exon 3 mutation encoding a Gly(137)Ser substitution was identified in a second CMT1 family. Each predicted amino acid substitution resides in the extracellular domain of the Po protein. Heteroduplex analysis did not detect either base change in 104 unrelated controls, indicating that these substitutions are disease-associated mutations rather than common polymorphisms. In addition, two polymorphic mutations were identified in MPZ exon 5 and exon 6, which do not alter the codons for Gly(200) and Ser(228), respectively. These observations provide further confirmation of the role of MPZ in CMT1B and suggest that MPZ coding region mutations may account for a limited percentage of disease-causing mutations in nonduplication CMT1 patients.

A 1.5-Mb deletion in 17p11.2-p12 is frequently observed in Italian families with hereditary neuropathy with liability to pressure palsies.

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder characterized by recurrent mononeuropathies. A 1.5-Mb deletion in chromosome 17p11.2-p12 has been associated with HNPP. Duplication of the same 1.5-Mb region is known to be associated with Charcot-Marie-Tooth disease type 1 (CMT1A), a more severe peripheral neuropathy characterized by symmetrically slowed nerve conduction velocity (NCV). The CMT1A duplication and HNPP deletion appear to be the reciprocal products of a recombination event involving a repeat element (CMT1A-REP) that flanks the 1.5-Mb region involved in the duplication/deletion. Patients from nine unrelated Italian families who were diagnosed with HNPP on the basis of clinical, electrophysiological, and histological evaluations were analyzed by molecular methods for DNA deletion on chromosome 17p. In all nine families, Southern analysis using a CMT1A-REP probe detected a reduced hybridization signal of a 6.0-kb EcoRI fragment mapping within the distal CMT1A-REP, indicating deletion of one copy of CMT1A-REP in these HNPP patients. Families were also typed with a polymorphic (CA)n repeat and with RFLPs corresponding to loci D17S122, D17S125, and D17S61, which all map within the deleted region. Lack of allelic transmission from affected parent to affected offspring was observed in four informative families, providing an independent indication for deletion. Furthermore, pulsed-field gel electrophoresis analysis of SacII-digested genomic DNA detected junction fragments specific to the 1.5-Mb HNPP deletion in seven of nine Italian families included in this study. These findings suggest that a 1.5-Mb deletion on 17p11.2-p12 is the most common mutation associated with HNPP.

Two autosomal dominant neuropathies result from reciprocal DNA duplication/deletion of a region on chromosome 17.

Charcot-Marie-Tooth disease type 1A (CMT1A) is a common autosomal dominant demyelinating neuropathy that is associated with a 1.5 megabase (Mb) tandem DNA duplication in chromosome 17p11.2-p12. Hereditary neuropathy with liability to pressure palsies (HNPP, tomaculous neuropathy) is another less frequently diagnosed autosomal dominant neuropathy and is associated with a 1.5 Mb deletion in chromosome 17p11.2-12. Meiotic unequal crossover is a proposed mechanism for the generation of both the duplication in CMT1A and the deletion in HNPP. CMT1A-REP is a repeat that flanks the region which is duplicated/deleted in CMT1A/HNPP. The CMT1A-REP repeat sequence may mediate unequal crossover through misalignment of the homologous, repeated sequences. Three copies of the CMT1A-REP repeat are present on stably inherited CMT1A duplication chromosomes. In this report, molecular analysis in multiple patients detected three copies of the CMT1A-REP sequence on both inherited and de novo CMT1A duplication chromosomes, and one copy of the CMT1A-REP repeat on the deleted chromosome in both inherited and de novo HNPP. These observations support the hypothesis that a reciprocal recombination mechanism involving the CMT1A-REP is responsible for the generation of both the duplicated and deleted chromosomes, and document the first examples in humans of Mendelian syndromes resulting from the reciprocal products of unequal exchange involving large intra-chromosomal segments.

Dejerine-Sottas syndrome associated with point mutation in the peripheral myelin protein 22 (PMP22) gene.

Dejerine-Sottas syndrome is a hypertrophic, demyelinating neuropathy which appears to demonstrate autosomal recessive inheritance in most pedigrees. Clinical symptoms are similar but more severe than Charcot-Marie-Tooth disease type 1 (CMT1), of which the major subtype, CMT1A, results either from duplication of a 1.5-megabase DNA region in chromosome 17p11.2-p12 containing the myelin gene PMP22, or from PMP22 point mutation. Mutational analysis of the PMP22 coding region in two unrelated Dejerine-Sottas patients identified individual missense point mutations present in the heterozygous state. These findings suggest that Dejerine-Sottas syndrome can result from dominant point mutation alleles of PMP22.

Evidence for a recessive PMP22 point mutation in Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is an autosomal dominant neuropathy that can be caused by dominant point mutations in PMP22 which encodes a peripheral nerve myelin protein. Usually, CMT1A is caused by the duplication of a 1.5-megabase (Mb) region on chromosome 17p11.2-p12 containing PMP22. Deletion of a similar 1.5-Mb region is associated with hereditary neuropathy with liability to pressure palsies (HNPP), a clinically distinct neuropathy. We have identified a severely affected CMT1 patient who is a compound heterozygote for a recessive PMP22 point mutation, and a 1.5 Mb deletion in 17p11.2-p12. A son heterozygous for the PMP22 point mutation had no signs of neuropathy, while two others heterozygous for the deletion had HNPP, suggesting that point mutations in PMP22 can result in dominant and recessive alleles contributing to CMT1A.