PMC8577214_01
Molecular genetic analyses of a young European American subject with a mutant C4B gene (MS630). Experimental approaches were similar to those described in 
Figure 3
. (A)
TaqI RFLP of three consecutive subjects to show RCCX structures. (B)
PshAI/PvuII to show the relative dosage of C4A and C4B. Notice that one C4B gene and three C4A genes were present in MS630 and MS632. (C) Immunofixation of C4A and C4B protein allotypes using EDTA-plasma from the same subjects shown in panels (A, B). No C4B was detectable in MS630. (D) DNA sequences at exon 17 from a control subject (upper panel) and MS630 (middle panel) showing a single C-nucleotide deletion in codon 755 (Q755) of C4B in MS630. The lower panel shows a C T polymorphism for codon 806 from exon 20 that did not change the amino acid sequence Val-806. (E)
EcoNI RFLP to detect the C-nucleotide deletion for MS630 and control M4. A 391-bp DNA fragment spanning intron 16 (I-16.5) and the 5' region of exon 18 (E18-r) was amplified by PCR, digested with EcoNI and resolved with agarose gel electrophoresis. Panel
(F) shows results of immunofixation (left) and immunoblot (right) experiments for five subjects. C4 protein allotypes in MS630 did not react with anti-Chido monoclonal. The GenBank accession number for the DNA fragment containing sequence with C-deletion in MS630 is NZ203455.
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  Figure 5E  showed immunofixation results for C4 protein allotypes for five members of the E94 family.
A European family with multiplex SLE-related mortality and low C4 levels (E94). (A) A three-generation family tree. E94P and S4 passed away with lupus nephritis and complications. (B)
TaqI RFLP to show RCCX modular structures for six subjects of the E94 family including the index patient E94P, two siblings (S1 and S2), three niece/nephew plus two control subjects (C01 and C02). Note that E94P, S1 and N3 each contained an unusual short-short (SS) haplotype for RCCX that had CYP21B-CYP21B configurations (
Table 2
). (C)
PshAI/PvuII RFLP to show the relative dosages of C4A and C4B genes for subjects shown in panel
(B), except E94P as her C4A and C4B genes were determined by real-time PCR. (D) Long-range mapping of the RCCX haplotypes by PmeI-digested genomic DNA in agarose plugs, resolved by pulsed-field gel electrophoresis and hybridized to radioactive probes specific for RCCX genes. DNA plugs for four members of the E94 family were prepared together with four unrelated controls (C03-C07). Subject N3 possessed the LL and SS haplotypes. (E) Immunofixation of EDTA-plasma to show C4A and C4B protein variants and their relative expression levels. Subject N3 has similar protein levels of C4A3 and C4B1, although she had LL/SS haplotypes with three copies of C4B gene and one copy of C4A. (F) DNA sequences at exon 28-intron 28 splice junction for six members of the E94 family, plus a C4 complete deficiency subject with identical HLA haplotypes (C4DF1), and a normal control (GN3104). Subjects S1, P and N3 had double sequences at the intron 28 splice junction donor site (marked by an arrow for E94P). C4DF1 has homozygous mutations at the same locations.
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The main clinical laboratory results were as follows: anti-nuclear antibody (ANA) titer very high at 1:2,560; anti-double stranded DNA detectable at 1:40; anti-SSA (Ro) positive; serum C3 very low at 35.5 mg/dL; C4 very low at 6.4 mg/dL ( Figure 5A ). Despite intensified immunosuppressive therapy, her disease progressed to central nervous system vasculitis with intracranial hemorrhage, which became lethal (family pedigree shown in  Figure 5A ). Limited quantity of genomic DNA from the index patient (E94P), plus blood samples from two of her siblings S1 and S2, two nieces N1 and N3 and a nephew N2 were available for studies of complement C4 genetic diversity ( Figure 5A ). On examining the constituents of the RCCX modules, the patient (E94P), her sibling S1 and her niece N3 shared a distinct haplotype characterized by the presence of two short C4 genes (SS) accompanied by two functional CYP21B genes (21B-21B; 3.7 kb;  Figure 5B ). N1 each showed a bimodular LL haplotype containing two mutant CYP21A genes (21A-21A; 3.2 kb;  Figure 5B ). N2 had regular constituents with one pseudogene and one functional gene for CYP21-hydroxylase, i.e., 21A-21B in each of his bimodular RCCX haplotypes. S2 and N2, heterozygous LL/LS for N1, and LL/SS for N3. Subjects C03 to C07 in  Figure 5D  were control samples with defined monomodular, bimodular and trimodular RCCX structures with different combinations of long and short genes (C03: LL/LS; C04: LL/LL; C05: LSS/LL; C06: LL/LS; C07: LS/L).   Figure 5E  showed immunofixation results for C4 protein allotypes for five members of the E94 family. T to A mutation at the donor site of intron 28 (GT AT). Thus, DNA fragments spanning exon 28 and intron 28 of C4 genes for six family members, E94P, S1, S2, N1, N2, N3, a completely C4-deficient patient (C4DF1) who was homozygous with this haplotype, plus an unrelated control (GM3104) were amplified by PCR and sequenced by Sanger's method.  Figure 5F  highlighted the sequence variations at the exon 28-intron 28 splice junction for those eight subjects.
PMC8577214_02
Genetic diversities of complement C4 - concepts and strategies of determination. (A) A simplified genetic map of the human leukocyte antigen (HLA) on the short arm of chromosome 6 highlighting genes relevant to immune response and inflammation. (B) Gene copy number variations (CNVs) of the RP-C4-CYP21-TNX (RCCX) modules. Haplotypes with one to five RCCX modules are shown. Blue, horizontal arrows above genes represent gene orientations and directions of transcription. The boxed structure at the right corner represents an RCCX module that is being duplicated. (C) Exon-intron structures with dichotomous size variation of long and short C4 genes. Symbols over exons depict locations encoding for the thioester bond (exon 24), C4A and C4B isotypic residues (exon 26), and major Rodgers and Chido blood group antigens (exon 28). An inverted arrow under intron 9 of the short C4 gene indicates the corresponding integration site for the endogenous retrovirus HERV-K(C4) in the long genes. (D) Differentiation of C4A and C4B genes and protein isotypes. Amino acid residues specific for C4A are shown in red fonts; amino acid residues specific for C4B are shown in blue fonts. PshAI restriction enzyme cleavage site specific for C4A is boxed. (E) A molecular map of gene organizations of the MHC-complement gene cluster with a bimodular long-short (LS) RCCX structure. Horizontal arrows stand for transcriptional orientations. The two PmeI cleavage sites that encompass the entire RCCX modules in pulsed field gel electrophoresis (PFGE) are indicated. (F) Structures of fourteen known RCCX length variants. The PmeI restriction fragment sizes resolved by PFGE are listed. Detailed experimental protocols for RCCX genotyping and C4 variants can be found in reference. The fragment sizes of TaqI restriction fragments for the RP-C4, CYP21 and TNX are shown on the right panel. Note that some individuals may contain duplicated copies of functional CYP21B (renamed to CYP21A2), or rearranged TNXA.
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Alleles for the more remote HLA-A ( Figure 1A ) exhibited diversity and three shared subtypes were found: *33:03, *24:02 and *11:01/02.
Identification of the molecular defect leading to C4B deficiency in an Asian patient with anti-NMDA receptor encephalitis (E133P). (A) Immunofixation of EDTA-plasma for C4 protein of the E133 patient (P), parents (M and F), and step-brother (S) plus an unrelated individual (UC). (B) Genomic DNA sequences showing conversion of W660 codon TGG conversion to nonsense codon TAG (W660x, upper). Middle and lower panels show two additional sequence variations for V825V from exon 20 (no change in amino acid) and for T907A from exon 21. (C) Genomic sequence that caused W660x mutation was distinguishable by AccI RFLP. (D) Analyses of the E133 family and control subjects. An 1.5 kb DNA fragment spanning between exon 14 to exon 18 from eight subjects were amplified by PCR, digested with restriction enzyme AccI and resolved by agarose gel electrophoresis. The results revealed that the W660x mutation was also present in his mother (E133M) and his stepbrother (E133S). (E) An improved method to detect W660x using a 0.6 kb fragment from intron 14 to intron 16 for AccI RFLP. Subject A285 was performed in duplicate to confirm the presence of W660x. The molecular weight marker was the kb-ladder. The GenBank accession number for the DNA fragment containing sequence with W660x from E133P is NZ203456.
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However, no C4B protein was detectable by immunofixation experiment (lane 1,  Figure 6A ) and thus patient E133P carried a mutant C4B gene. The patient's father had homozygous, monomodular-long RCCX (L/L) haplotypes with two copies of C4 genes both coding for C4A protein (lane 3,  Figure 6A ) and thus also had a homozygous C4B deficiency. TAG, W660x) at exon 15 ( Figure 6B ). The DNA mutation for W660x created a new cleavage site for the AccI restriction enzyme (GTAGAC,  Figure 6C ). AccI digest and resolved by agarose gel electrophoresis ( Figure 6D ). AccI digested fragments of 0.9 kb and 0.6 kb were present in the patient (E133P), his mother (E133M) and maternal stepbrother (E133S), but not in his father (E133F) and four unrelated controls. The W660x nonsense mutation was firstly detected in an independent East-Asian healthy subject, A-285, residing in the US (panel E,  Figure 6 ).