Human Mutation

D’Elia, Angela V.; Tell, Gianluca; Paron, Igor; Pellizzari, Lucia; Lonigro, Renata; Damante, Giuseppe

doi: 10.1002/humu.1207pmid: 11668629

The homeodomain (encoded by the homeobox) is the DNA‐binding domain of a large variety of transcriptional regulators involved in controlling cell fate decisions and development. Mutations of homeobox‐containing genes cause several diseases in humans. A variety of missense mutations giving rise to human diseases have been described. These mutations are an excellent model to better understand homeodomain molecular functions. To this end, homeobox missense mutations giving rise to human diseases are reviewed. Seventy‐four independent homeobox mutations have been observed in 17 different genes. In the same genes, 30 missense mutations outside the homeobox have been observed, indicating that the homeodomain is more easily affected by single amino acids changes than the rest of the protein. Most missense mutations have dominant effects. Several data indicate that dominance is mostly due to haploinsufficiency. Among proteins having the homeodomain as the only DNA‐binding domain, three “hot spot” regions can be delineated: 1) at codon encoding for Arg5; 2) at codon encoding for Arg31; and 3) at codons encoding for amino acids of recognition helix. In the latter, mutations at codons encoding for Arg residues at positions 52 and 53 are prevalent. In the recognition helix, Arg residues at positions 52 and 53 establish contacts with phosphates in the DNA backbone. Missense mutations of amino acids that contribute to sequence discrimination (such as those at positions 50 and 54) are present only in a minority of cases. Similar data have been obtained when missense mutations of proteins possessing an additional DNA‐binding domain have been analyzed. The only exception is observed in the POU1F1 (PIT1) homeodomain, in which Arg58 is a “hot spot” for mutations, but is not involved in DNA recognition. Hum Mutat 18:361–374, 2001. © 2001 Wiley‐Liss, Inc.

Hymes, Jeanne; Stanley, Christine M.; Wolf, Barry

doi: 10.1002/humu.1208pmid: 11668630

Biotinidase (BTD) is the only enzyme that can cleave biocytin, a product of the proteolytic digestion of holocarboxylases. Profound BTD deficiency (less than 10% mean normal activity in serum) is an autosomal recessive disorder that can result in neurological and cutaneous abnormalities. Both the cDNA and the genomic DNA of normal BTD gene have been isolated and characterized. The BTD gene is localized to chromosome 3p25. Thus far 61 mutations in three of the four exons of the BTD and one mutation in an intron gene that cause profound BTD deficiency have been reported. Mutations occur at different frequencies in symptomatic children than they do in children ascertained by newborn screening. Two mutations, 98‐104del7ins3 and R538C, were present in 52% or 31 of 60 alleles found in symptomatic patients. Three other mutations, A755G, Q456H, and 511 G>A; 1330G>C (double mutation), accounted for 52% of the alleles detected by newborn screening in the United States. Two asymptomatic adults, parents of children with profound BTD deficiency detected by newborn screening, have been described. Additional different mutations have been found in Turkish, Saudi Arabian, and Japanese children with profound BTD deficiency. Partial BTD deficiency (10–30% of mean normal serum activity) is predominantly caused by the single 1330G>C mutation that results in D444H on one allele in combination with one of the mutations causing profound deficiency on the other allele. Four intragenic polymorphisms, three neutral and one amino acid change, have also been found. Although a preponderance of mutations causing the production of truncated BTD protein occurs in symptomatic children with profound deficiency, preliminary studies fail to demonstrate clear genotype–phenotype correlations. Hum Mutat 18:375–381, 2001. © 2001 Wiley‐Liss, Inc.

Zhang, Ge; Zhang, Sizhong; Chen, Wei; Qiu, Weimin; Wu, Hui; Wang, Jianmin; Luo, Jingchu; Gu, Xiaocheng; Cotton, Richard G.H.

doi: 10.1002/humu.1209pmid: 11668631

Human genome polymorphisms play a key role in defining the molecular basis of phenotypic differences between individuals in aspects such as disease susceptibility and drug responses. The database requirements for supporting the study of human genetic variation have been well recognized. In order to meet these needs, several generalized databases have been built. However, it is still hard for users to find gene‐related variation data from these huge and sophisticated databases. In its role as a gene‐oriented directory of polymorphism data, Go!Poly (Gene Oriented Polymorphism Database; http://61.139.84.5/gopoly/) utilizes two new highly curated and non‐redundant resources, LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/) and RefSeq (http://www.ncbi.nlm.nih.gov/LocusLink/refseq.html), as the standard for identifying and positioning nucleotide variations. As a generalized polymorphism database, Go!Poly extracts human gene‐linked sequence variations of all common types (SNP, insertion‐deletion, simple tandem repeat, and complex nucleotides variations) from various public resources including scientific journals and internet resources, such as HGBASE (http://hgbase.cgr.ki.se) and dbSNP (http://www.ncbi.nlm.nih.gov/SNP/). The polymorphism data are then categorized into different gene loci, and the reference sequences given by LocusLink are used as positioning references. Through close integration with LocusLink, Go!Poly also provides facilitated connections among sequence data, gene name, and related biological information. This feature also makes Go!Poly easy to search and navigate. Future automated annotations and internal consistency checking may also benefit from this. Extensive efforts are being taken to make the polymorphism information generated by the Chinese scientific community available from this resource. Hum Mutat 18:382–387, 2001. © 2001 Wiley‐Liss, Inc.

Goudeau, Bertrand; Dagvadorj, Ayush; Rodrigues‐Lima, Fernando; Nédellec, Patrick; Casteras‐Simon, Monique; Perret, Emmanuelle; Langlois, Sylvie; Goldfarb, Lev; Vicart, Patrick

doi: 10.1002/humu.1210pmid: 11668632

Desmin‐related myopathy is a familial or sporadic disease characterized by skeletal muscle weakness and cardiomyopathy as well as the presence of intracytoplasmic aggregates of desmin‐reactive material in the muscle cells. Previously, two kinds of deletions and eight missense mutations have been identified in the desmin gene and proven to be responsible for the disorder. The present study was conducted to determine structural and functional defects in a pathogenic desmin variant that caused a disabling disorder in an isolated case presenting with distal and proximal limb muscle weakness and cardiomyopathy. We identified a novel heterozygous Q389P desmin mutation located at the C‐terminal part of the rod domain as the causative mutation in this case. Transfection of desmin cDNA containing the patient’s mutation into C2.7, MCF7, and SW13 cells demonstrated that the Q389P mutant is incapable of constructing a functional intermediate filament network and has a dominant negative effect on filament formation. We conclude that Q389P mutation is the molecular event leading to the development of desmin‐related myopathy. Hum Mutat 18:388–396, 2001. © 2001 Wiley‐Liss, Inc.

Abed, Abdalla A.; Günther, Klaus; Kraus, Cornelia; Hohenberger, Werner; Ballhausen, Wolfgang G.

doi: 10.1002/humu.1211pmid: 11668633

This study was intended to evaluate a diagnostic reverse transcriptase polymerase chain reaction based protein‐truncation test for the identification of germline mutations in the serine/threonine protein kinase 11 (STK11, also designated LKB1) gene in Peutz‐Jeghers syndrome (PJS). Our data exemplify that the inactivation of STK11 can be due to unusual disturbances in splicing regulation which result in truncations of the protein. However, nonsense mediated mRNA decay must be blocked with puromycin to detect shortened STK11 gene products contained in the leucocytic mRNA pool of PJS patients. Interestingly, two mutations escaped from detection by exon sequencing techniques with usual flanking PCR primers, since alterations were located right in the middle of intronic sequences. We describe a compound heterozygous PJS patient who carried two different mutations in intron 1 on separate alleles. Each of the two mutations was transmitted individually to one of his two children. In the course of our RNA based analyses we detected high level expression of a novel STK11/LKB1 mRNA variant retaining intron 4 (STK11 c.597⁁598insIVS4) in various tissues. This mRNA isoform was initiated from an alternative transcription regulatory region as revealed by primer extension analyses even in cell lines with complete methylation of the normal promoter. As a consequence of novel mutational mechanisms identified we discuss the impact of RNA based strategies for the detection of germinal STK11 mutations in PJS. Hum Mutat 18:397–410, 2001. © 2001 Wiley‐Liss, Inc.

D’Souza‐Li, Lilia; Canaff, Lucie; Janicic, Natasa; Cole, David E.C.; Hendy, Geoffrey N.

doi: 10.1002/humu.1212pmid: 11668634

We studied family members of a large kindred expressing both familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) and found, by PCR amplification of the extracellular calcium‐sensing receptor (CASR) gene exons and flanking intronic sequences, that FHH individuals were heterozygous for a g to t substitution in the last nucleotide of intron 2 (IVS2‐1G>T). Defects in messenger RNA splicing were investigated by illegitimate transcription of the CASR gene in lymphoblastoid cells from an FHH affected individual, as well as by transfection of a CASR minigene harboring this mutation into HEK293 cells. The mutation resulted predominantly in exon III skipping causing a shift in exon IV reading frame and introduction of a premature stop codon leading to a predicted truncated protein of 153 amino acids. Interestingly, it was noted that exon III splicing is not 100% efficient in parathyroid, thyroid, and kidney; an exon III‐deleted transcript is produced approximately 15% of the time. This is the first description of a splice site mutation in the CASR gene and provides an explanation of the clinical phenotype of the patients. Hum Mutat 18:411–421, 2001. © 2001 Wiley‐Liss, Inc.

Watanabe, Yoriko; Murray, Jeffrey C.; Bjork, Bryan C.; Bird, Christine P.; Chiang, P.‐W.; Gregory, Simon G.; Kurnit, David M.; Schutte, Brian C.

doi: 10.1002/humu.1213pmid: 11668635

Van der Woude syndrome (VWS) is an orofacial clefting disorder with an autosomal dominant pattern of inheritance. In our efforts to clone the VWS gene, 900 kb of genomic sequence from the VWS candidate region at chromosome 1q32‐q41 was analyzed for new DNA sequence variants. We observed that in clone CTA‐321i20 a 7922 bp sequence is absent relative to the sequence present in PAC clone RP4‐782d21 at positions 1669‐9590, suggesting the presence of a deletion/insertion (del/ins) polymorphism. Embedded in this 7922 bp region was a TTCC short tandem repeat (STR). Genotype analysis showed that both the internal STR and the (del/ins) mutation were true polymorphisms. This is a novel example of intraallelic variation, a polymorphism within a polymorphism, and we suggest that it be termed a “Matroshka” polymorphism. Further genetic and DNA sequence analysis indicated that the ancestral state of the 1669‐9590 del/ins polymorphism was the insertion allele and that the original deletion mutation probably occurred only once. A second class of novel DNA sequence variation was discovered on chromosome 5 that shared a 328 bp identical sequence with this region on chromosome 1. A single nucleotide polymorphism (SNP) was detected by SSCP using a pair of primers derived from the chromosome 1 sequence. Surprisingly, these primers also amplified the identical locus on chromosome 5, and the SNP was only located on chromosome 5. Since the probe unexpectedly detected alleles from another locus, we suggest that this type of sequence variant be termed an “ectopic” polymorphism. These two novel classes of DNA sequence polymorphisms have the potential to confound genetic and DNA sequence analysis and may also contribute to variation in disease phenotypes. Hum Mutat 18:422–434, 2001. © 2001 Wiley‐Liss, Inc.

Mizunuma, Makiko; Fujimori, Shin; Ogino, Hitoshi; Ueno, Takamasa; Inoue, Hirokazu; Kamatani, Naoyuki

doi: 10.1002/humu.1214pmid: 11668636

We identified the identical large genomic deletion in the hypoxanthine phosphoribosyltransferase (HPRT1) gene in two Japanese patients with Lesch‐Nyhan (LN) syndrome. This deletion spanned from an Alu sequence in the promoter region to another Alu‐sequence in intron 1, a length of 2,969 base pairs including exon 1. In order to ask whether this deletion was a recurrent mutation, we developed a simple alternative method to determine the separate origin of the HPRT1 mutation of the patients as assessed with an apparent mtDNA polymorphism. Considering that an LN syndrome‐causing mutation is not transmitted from patient to offspring as LN syndrome is a fatal disease in childhood and that mtDNA is maternally inherited, HPRT1 mutations and mtDNA would be co‐transmitted from carrier mother to offspring since both appeared in females. Two bases were different in the hypervariable region I of the mtDNA between the two patients, indicating the separate origin of their mtDNA over at least several thousand years as calculated based on the molecular evolution rate in this region. We thus conclude that the identical deletion found in HPRT1 of the two patients was derived from recurrent events of genomic recombination. Given that the same Alu‐mediated deletion of HPRT1 has not been reported among somatic mutations at the same locus, this region of the HPRT1 gene flanked by Alu‐sequences is likely a mutational hot spot in the germline but not in somatic cells. In addition, we also report novel LN‐syndrome‐conferring mutations in intron 6 (IVS6+1G → C) and intron 8 (IVS7‐9T → G) that resulted in exclusions of exon 6 and exon 8, respectively. Hum Mutat 18:435–443, 2001. © 2001 Wiley‐Liss, Inc.

Gelhaus, Annette; Scheding, Andreas; Browne, Edmund; Burchard, Gerd D.; Horstmann, Rolf D.

doi: 10.1002/humu.1215pmid: 11668637

Studying 12 selected individuals from a malaria‐endemic area in West Africa, 24 variants of the CD36 gene were found, 21 of them novel ones. These included three single‐nucleotide substitutions causing non‐conservative amino acid exchanges E123K, T174A, and I271T as well as a three base pair (bp) insertion resulting in the addition of an asparagine residue (N232‐233ins). The E123K variant was located within the putative ligand‐binding domain for oxidized low density lipoprotein, while the other substitutions resided outside any of the binding sites for reaction partners mapped on CD36 so far. Twelve single‐nucleotide polymorphisms (SNPs) were identified in untranslated parts of the exons and in introns. Five additional SNPs were located in the promoter region whereby ‐144G→T, ‐53G→T, and ‐2A→G alter putative binding sites for the transcription factors purine factor (PuF), phorbol ester‐responsive element AP‐2, and CCAAT/enhancer‐binding protein. A G→T exchange at position –50 appears to introduce a new recognition site for PuF. Calculations of nucleotide diversity revealed extraordinarily high numbers for all parts of the gene, which may, however, to some extent be due to the selection of individuals studied. Hum Mutat 18:444–450, 2001. © 2001 Wiley‐Liss, Inc.

Larsen, Lars Allan; Johnson, Martin; Brown, Candia; Christiansen, Michael; Frank‐Hansen, Rune; Vuust, Jens; Andersen, Paal Skytt

doi: 10.1002/humu.1216pmid: 11668638

The rapid progress in the isolation of genes associated with human disease has resulted in an increasing demand for mutation screening methods. The molecular diagnosis of the long QT syndrome (LQTS), a cardiac disorder characterized by prolongation of the QTc interval in the ECG, syncopes, and sudden death, requires mutation screening of all exons in at least five genes, encoding cardiac Na+ and K+ channel subunits. A method for automated dideoxy fingerprinting (ddF) using capillary array electrophoresis (CAE) was developed and the efficiency of the method was tested by analyzing 24 DNA samples with mutations in one of the genes KCNQ1 and KCNH2, which are involved in 50% of LQTS cases. One of these mutations, 362insQK in KCNQ1, is novel. The sensitivity was 100% using a single electrophoresis temperature of 18°C or 25°C. However, analysis of the samples in both the sense and anti‐sense direction were required for high sensitivity. Analysis in a single direction resulted in a decrease of the sensitivity to 74% and 70%, respectively. The throughput of the ddF method, if performed with a 16 capillary CAE instrument, is 288 samples per seven hr if each sample is analyzed on both strands. Hum Mutat 18:451–457, 2001. © 2001 Wiley‐Liss, Inc.