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Objectives. To determine the utility of single-stranded conformation polymorphism (SSCP) analysis for mutation screening in the BTK (Bruton's tyrosine kinase) gene, we investigated 56 X-linked agammaglobulinemia (XLA) families. To obtain genotype/phenotype correlations, predicted protein aberrations were correlated with the clinical course of the disease.Patients. This study included 56 patients with XLA, with or without a positive family history, who were diagnosed on the basis of their clinical features, low peripheral B-cell count, and low immunoglobulin levels. Ten patients with isolated hypogammaglobulinemia and 50 healthy males served as controls.Methods. SSCP analysis was performed for the entire BTK gene, including the exon–intron boundaries and the promoter region. Structural implications of the missense mutations were investigated by molecular modeling, and the functional consequences of some mutations also were evaluated by in vitro kinase assays and Western blot analysis.Results. We report the largest series of patients with XLA to date. All but 5 of the 56 index patients with XLA screened with SSCP analysis showed BTK gene abnormalities, and in 2 of the 5 SSCP-negative patients, no BTK protein was found by Western blot analysis. There were 51 mutations, including 37 novel ones, distributed across the entire gene. This report contains the first promoter mutation as well as 14 novel missense mutations with the first ones described for the Tec homology domain and the glycine-rich motif in the SH1 domain. Each index patient had a different mutation, except for four mutations, each in two unrelated individuals. This result supports the strong tendency for private mutations in this disease. No mutations were found in the controls.Conclusions. Our results demonstrate that molecular genetic testing by SSCP analysis provides an accurate tool for the definitive diagnosis of XLA and the discrimination of borderline cases, such as certain hypogammaglobulinemia or common variable immunodeficiency patients with overlapping clinical features. Genotype/phenotype correlations are not currently possible, making prediction of the clinical course based on molecular genetic data infeasible.

A nonspecific X-linked mental retardation (MRX) family is reported with four mild to moderately affected males and no intellectual impairment in their obligate carrier mothers. Linkage analysis obtained the same multipoint lod score of 2.08 for two intervals on the X chromosome already reported to be linked to other MRX and syndromic X-linked mental retardation (XLMR) families: one pericentromeric and the other at Xq26. Since the responsible gene is not yet characterized, haplotyping is presently the only means available for carrier and prenatal testing for this form of MRX. Carrier risk estimation using pedigree and haplotype data for five females at risk is presented, and the difficulties of prenatal diagnosis given linkage to two different regions is discussed.

In the search for genetic causes of mental retardation, we have studied a five-generation family that includes 10 individuals in generations IV and V who are affected with mild-to-moderate mental retardation and mild, nonspecific dysmorphic features. The disease is inherited in a seemingly autosomal dominant fashion with reduced penetrance. The pedigree is unusual because of (1) its size and (2) the fact that individuals with the disease appear only in the last two generations, which is suggestive of anticipation. Standard clinical and laboratory screening protocols and extended cytogenetic analysis, including the use of high-resolution karyotyping and multiplex FISH (M-FISH), could not reveal the cause of the mental retardation. Therefore, a whole-genome scan was performed, by linkage analysis, with microsatellite markers. The phenotype was linked to chromosome 16p13.3, and, unexpectedly, a deletion of a part of 16pter was demonstrated in patients, similar to the deletion observed in patients with ATR-16 syndrome. Subsequent FISH analysis demonstrated that patients inherited a duplication of terminal 3q in addition to the deletion of 16p. FISH analysis of obligate carriers revealed that a balanced translocation between the terminal parts of 16p and 3q segregated in this family. This case reinforces the role of cryptic (cytogenetically invisible) subtelomeric translocations in mental retardation, which is estimated by others to be implicated in 5%10% of cases.

The gene for ubiquitin hydrolase on the X chromosome (UHX1), cloned and mapped to Xp21.2-p11.2, is a candidate gene for retinal diseases. We used fine mapping techniques to localise UHX1 between markers DXS1266 and DXS337, where congenital stationary night blindness (XICSNB) and retinitis pigmentosa type 2 (RP2) are also located. Reevaluation of the UHX1 gene structure demonstrated five new exons, for a total of 21 exons and a predicted protein product of 963 amino acids. Evaluation of patients revealed no UHX1 mutations using SSCP (10 CSNB1 and 20 XLRP) or deletion screening with cDNA hybridisation (13 CSNB1 and 43 XLRP). Likewise, no aberrations were found in the nearby PCTAIRE1 (PCTK1) gene in 13 CSNB1 and 43 XLRP patients by deletion screening. Thus mutations of UHX1, and probably PCTK1, do not appear to cause common X-linked eye diseases. UHX1's role in patients with mental retardation may be appropriate for further investigations into UHX1 function.

We report on 15 patients with velo-cardiofacial syndrome who had a severe form of tetralogy of Fallot (pulmonary atresia, ventricular septal defect, and hypoplastic pulmonary arteries). Noncardiac anomalies in these patients included typical facial and ear anomalies in 15, nasal speech in 13, palate anomalies in 10, and developmental delay in 10. Seven patients had significant bronchospasm, which has not been reported in association with the velo-cardio-facial syndrome. All 15 patients had severe abnormalities of the arborization of the pulmonary arterial tree, which also has not been reported in velo-cardio-facial syndrome. All patients underwent staging operations to prepare the true pulmonary vascular tree for complete repair of the defect (five underwent complete repair and three survived). Of the remaining 10 patients, 6 are awaiting further operation, 3 are not candidates for complete repair, and 1 has died.

The Schimmelpenning-Feuerstein-Mims syndrome (SFM), characterized by linear nevus sebaceous and ocular and neurologic abnormalities, is a sporadic condition without known familial cases or etiology. We report the occurrence of SFM in only one of two monozygotic (MZ) twins. After considering a variety of possible causative mechanisms, we suggest that a postzygotic dominant lethal mutation in mosaic form may best explain SFM and the discordancy for SFM in these MZ twins.

COX deficiency is believed to be the most common defect in neonates and infants with mitochondrial diseases. To explore the causes of this group of disorders, we examined 25 mitochondrial genes (three COX subunit genes and 22 tRNA genes) and 10 nuclear COX subunit genes for disease associated mutations using PCR-SSCP and direct sequencing of polymorphic SSCP fragments. DNA from one patient with severe COX deficiency and with consanguineous parents was entirely sequenced. The patient population consisted of 21 unrelated index patients with mitochondrial disorders and predominant (n=7) or isolated (n=14) COX deficiency. We detected two distinct tRNA(Ser)(UCN) mutations, which have been recently described in single kindreds, in a subgroup of four patients with COX deficiency, deafness, myoclonic epilepsy, ataxia, and mental retardation. Besides a number of nucleotide variants, a single novel missense mutation, which may contribute to the disease phenotype, was found in the mitochondrial encoded COX 1 gene (G6480A). Mutations in nuclear encoded COX subunit genes were not detected in this study.